CN1832762B - Conjugates of hydroxyalkyl starch and g-csf - Google Patents

Abstract

The present invention relates to conjugates of hydroxyalkyl starch and a granulocyte colony stimulating factor protein (G-CSF) wherein these conjugates are formed by a covalent linkage between the hydroxyalkyl starch or a derivative of the hydroxyalkyl starch and the protein. The present invention also relates to the methods of producing these conjugates and the use of these conjugates.

Description

Conjugates of Hydroxyalkyl Starch and G-CSF

technical field

The invention relates to conjugates of hydroxyalkyl starch and granulocyte colony-stimulating factor protein (G-CSF), wherein the conjugates are formed by covalent bonds between hydroxyalkyl starch or derivatives of hydroxyalkyl starch and protein. The invention also relates to methods for the preparation of these conjugates, and the use of these conjugates. the

Background technique

It is generally known that when these proteins are coupled to polymeric molecules, the stability of the proteins is improved and the immune response against these proteins is reduced. WO 94/28024 reveals that the physiologically active protein modified by polyethylene glycol (PEG) has low immunogenicity and antigenicity, and the lifespan in blood circulation is significantly higher than that of uncoupled protein, that is, the clearance rate is reduced. the

G-CSF is a 21 kDa glycoprotein that is stabilized by two intrachain disulfide bonds and contains a single O-linked carbohydrate moiety. Mature G-CSF has 174 amino acids. G-CSF in animals is synthesized by bone marrow stromal cells, macrophages and fibroblasts. Its main function is as a growth and differentiation factor for neutrophils and their precursors. However, it is also known in the art that G-CSF activates mature neutrophils. In addition, it stimulates the growth/differentiation of various other hematopoietic progenitor cells (in synergy with other hematopoietic growth factors) and promotes proliferation and migration of endothelial cells. Clinically, G-CSF is administered to treat deficiencies in neutrophil levels (eg, caused by aplastic anemia, myelodysplasia, AIDS or chemotherapy). the

WO 02/09766 discloses inter alia biocompatible protein-polymer compounds made by conjugating biologically active proteins with biocompatible polymer derivatives. The biocompatible polymer used is a highly reactive branched polymer and the resulting conjugate contains a long linker between the polymer derivative and the protein. As biocompatible polymers, polymers of the general formula (P- _OCH2CO -NH-CHR-CO-) _n -LQk- _A are described, where P and Q are polymeric residues and k can be 1 or 0. P and Q mentioned polyethylene glycol, polypropylene glycol, polyoxyethylene, polytrimethyleneglycol (polytrimethyleneglycol), polylactic acid and its derivatives, polyacrylic acid and its derivatives, polyamino acid, polyvinyl alcohol, Polyurethanes, polyphosphazenes, poly(L-lysine), polyalkylene oxides, polyacrylamides, and water-soluble polymers such as dextran or polysaccharides. Among the proteins are especially mentioned alpha-, beta- and gamma-interferons, blood factors, cytokines such as interleukins, G-CSF, GM-CSF. The examples of WO 02/09766 disclose only mono-, di- and tri-polyethylene glycol derivatives coupled only to interferon and epidermal growth factor and human growth hormone.

WO 94/01483 discloses biocompatible polymer conjugates formed by the covalent bonding of a biologically inactive polymer or polymer derivative to a pharmaceutically pure synthetic hydrophilic polymer via a specific type of chemical bond. The natural polymers and their derivatives disclosed therein are polysaccharides such as hyaluronic acid, proteoglycans such as chondroitin sulfate A, B and C, chitin, heparin, heparan sulfate, dextran such as cyclodextrin, hydroxyl Ethylcellulose, cellulose ethers and starches, lipids such as triglycerides and phospholipids. Synthetic polymers mentioned are polyethylene and its derivatives having an average molecular weight of from about 100 to about 100,000. Proteins linked to polymers or polymer derivatives mentioned are cytokines and growth factors, including interferons, tumor necrosis factors, interleukins, colony-stimulating factors, growth factors such as osteogenic factor extract, epidermal growth factor, transforming growth factor, platelet-derived growth factor, acidic fibroblast growth factor, etc. All examples of WO 94/01483 use polyethylene glycol derivatives as polymers. the

WO 96/11953 discloses N-terminal chemically modified protein compounds and methods for their preparation. Specifically, G-CSF compositions formed by coupling a water-soluble polymer to the N-terminus of G-CSF are described therein. Combination interferon conjugated at the N-terminus to a water-soluble polymer is also disclosed in WO 96/11953. Although WO 96/11953 lists many kinds of water polymers (such as copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diox Acetane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol polymers, polyoxypropylene/ethylene oxide copolymers or polyoxyethylated polyols), but only PEGylated G-CSF or combined IFN compositions are described in the examples of WO 96/11953. the

The polypeptide conjugate disclosed in US 6,555,660B2 comprises a polypeptide having G-CSF activity, and its amino acid sequence differs from the human G-CSF amino acid sequence in that at least one specifically introduced and/or removed amino acid residue, wherein the The conjugate comprises a group linking a non-polypeptide moiety and further comprises at least one non-polypeptide moiety attached to the linking group of the polypeptide. The non-polypeptide moiety may be a polymer such as polyethylene glycol or an oligosaccharide. It is clearly stated in US 6,555,660B2 that PEG is currently the most preferred polymer molecule because it has only very few reactive groups that can be cross-linked compared with polysaccharides such as dextran. the

WO 97/30148 relates to polypeptide conjugates with reduced allergenicity comprising a polymeric carrier molecule coupled to two or more polypeptide molecules. These conjugates are preferably part of compositions used in the personal care market. The preparation method of the conjugate comprises: activating the polymer carrier molecule, reacting two or more polypeptide molecules with the activated polymer carrier molecule, and blocking the residual active group on the conjugate. A variety of polymeric carrier molecules are listed in WO 97/30148, including different classes of compounds such as natural or synthetic homopolymers, such as polyols, polyamines, polycarboxylic acids and heteropolymers comprising at least two different linking groups . Examples given include star PEGs, branched PEGs, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidone and poly-D,L-amino acids. It also discloses dextran, such as carboxymethyl dextran, cellulose, such as hydroxyethyl cellulose or hydroxypropyl cellulose, hydrolyzate of chitosan, starch, such as hydroxyethyl starch or hydroxypropyl Starch, glycogen, agarose, guar gum, inulin, pullulan, xanthan gum, carrageenan, pectin, alginic acid, etc. As for polypeptides, only a few enzymes are explicitly disclosed therein. the

Baldwin, J.E. et al., Tetrahedron, vol.27(1981), pp.1723-1726 describe the chemical modification of dextran with hydroxyethyl starch, the resulting aldehyde-substituted polymer can react with hemoglobin to produce soluble Hemoglobin bound to a polymer. It has been shown to bind oxygen, but cardiac perfusion experiments clearly demonstrate that this polymer-bound hemoglobin is not suitable for use as a blood substitute. the

WO 99/49897 describes hemoglobin conjugates formed by reacting polysaccharides such as dextran or hydroxyethyl starch with amino groups of hemoglobin. The aldehyde group generated by opening the sugar ring through oxidation reaction is used as the functional group of the polysaccharide. Borane dimethylamine is disclosed as a preferably used reducing agent. Furthermore, WO 99/49897 is limited to hemoglobin. the

WO 03/074087 relates to a method for coupling proteins to starch-derived modified polysaccharides. The binding effect between protein and polysaccharide (hydroxyalkyl starch) is a covalent connection. This covalent connection is between the terminal aldehyde group of the hydroxyalkyl starch molecule or the functional group obtained after chemical modification of the terminal aldehyde group and the functional group of the protein. formed between. Amino, thio, and carboxyl groups are disclosed as reactive groups of proteins, but aldehyde groups of proteins are not mentioned. Furthermore, although numerous possibilities for different linkages are listed therein, including different functional groups, different linker molecules that are theoretically suitable and different chemistries, the examples only give two options: first, the use of oxidized hydroxyethyl Starch, activated by ethyl dimethylaminopropyl carbodiimide (EDC), directly coupled to protein, or unoxidized hydroxyethyl starch, directly coupled to protein to form Schiff's base ), which are then reduced to the respective amines. Thus, the examples of WO 03/074087 neither disclose single conjugates coupled through the thio or carboxyl groups of the protein, nor describe conjugates comprising hetastarch, the protein and one or more linker molecules. In addition, G-CSF molecules were not used in the examples. the

Therefore, an object of the present invention is to provide a conjugate of hydroxyalkyl starch, preferably hydroxyethyl starch, and G-CSF which has not been described in the prior art. the

Another object of the present invention is to provide methods for the preparation of these conjugates. the

Contents of the invention

Therefore, the present invention relates to a method for preparing a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), and the protein is granulocyte colony stimulating factor (G-CSF ), the method comprising reacting at least one functional group A of a polymer or derivative thereof with at least one functional group Z of a protein, thereby forming a covalent linkage, wherein Z is selected from the group consisting of amino, thiol, aldehyde and ketone groups, and

- where, if Z is an aldehyde or ketone group, A comprises an amino group forming the linkage with Z, or

-wherein, if Z is amino, A is selected from reactive carboxyl and aldehyde, ketone or hemiacetal,

--wherein, if A is an aldehyde group, a ketone group or a hemiacetal group, the method also includes introducing A into the polymer to form a polymer derivative

---Reaction of a polymer with at least one bifunctional compound, one of which can react with the polymer, where at least another functional group is an aldehyde, ketone or hemiacetal, or can be further chemically modified to produce an aldehyde A functional group of a group, a keto group or a hemiacetal group, or

--- Oxidize the polymer to produce at least one, especially at least two aldehyde groups, or

--wherein, if A is a reactive carboxyl group, the method also includes introducing A into the polymer to form a polymer derivative

--- Selectively oxidize the reducing end of the polymer and activate the resulting carboxyl group, or

--- The non-redox end of the polymer is reacted with a carbonic acid diester, or

-wherein, if Z is a thiol group, A contains

--maleimide or

--Haloacetyl

Form the connection with Z. the

Therefore, the present invention also relates to conjugates that can be prepared using the above methods. the

G-CSF can be prepared by chemical synthesis, or can be derived from any human (see, e.g., Burgess, A.W. et al. 1977, Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells. Blood 49 (1977), 573-583; Shah, R.G. et al. 1977, Characterization of colony-stimulating activity produced by humanmonocytes and phytohemagglutinin-stimulated lymphocytes.Blood50 (1977), 811) or another mammalian source, and can be produced by such as human placenta, human blood or human urine, etc. Purified from natural sources. In addition, many epithelial carcinomas, acute myelogenous leukemia cells, and various tumor cell lines (bladder cancer, medulloblastoma) are capable of expressing this factor. the

Furthermore, the expression G-CSF also includes G-CSF variants in which one or more amino acids (eg 1 to 25, preferably 1 to 10, more preferably 1 to 5, most preferably 1 or 2) have been replaced by Substitution to another amino acid and exhibiting G-CSF activity (see for example: Riedhaar-Olson, J.F. et al. 1996, Identification of residue critical to the activity of human granulocyte colony-stimulating factor. Biochemistry 35: 9034-9041 1996; U.S. Pat. Nos. 5,581476; 5,214,132; 5,362,853; 4,904,584). Assays for G-CSF activity are described in the art (for in vitro assays of G-CSF activity see e.g.: Shirafuji, N. et al. 1989, A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders, Exp. Hematol. 1989, 17, 116-119; for in vivo assay of G-CSF activity see eg: Tanaka, H. et al. 1991, Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats, Cancer Research 51, 3710-3714, 1991). Other literatures on methods for measuring G-CSF activity are U.S. Patent No. 6,555,660; Nohynek, G.J. et al., 1997, Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factors inneutropenic and nonneutropenic CDr ol he rats. 1997) 39; 259-266. the

G-CSF is preferably produced recombinantly. It includes exogenous DNA sequences expressed by genomic or cDNA cloning or DNA synthesis from prokaryotic or eukaryotic hosts. Suitable prokaryotic hosts include various bacteria such as Escherichia coli (E. coli). Suitable eukaryotic hosts include yeast, such as S. cerevisiae, and mammalian cells, such as Chinese hamster ovary cells and monkey cells. the

Methods for the recombinant production of proteins are known in the art. Generally, it involves transfecting a host cell with an appropriate expression vector, culturing the host cell under conditions such that the protein is produced, and purifying the protein from the host cell. For details, see for example: Souza, L.M. et al. 1986, Recombinant human granulocyte colony-stimulating factors: effects on normal and leukemic myeloid cells. Science 1986232: 61-65, 1986; Nagata, S. et al. 1986, Molecular DNA cloning and expression of for human granulocyte colony-stimulating factors. Nature 319: 415-418, 1986; Komatsu, Y. et al. 1987, Cloning of human granulocyte colony-stimulating factors cDNA from human macrophages and its expression in Escherichia Rescoli, Jpn 8 Rescoli, 19 Cancer 7 J (19 Cancer 7 J) : 1179-1181. the

In a preferred embodiment, G-CSF has the amino acid sequence of human mature G-CSF (see for example: Nagata, S. et al. 1986, Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factors. Nature 319:415-418, 1986), and can further include a methionine at its amino terminus, resulting in a protein of 175 amino acids. In addition, G-CSF may contain serine or threonine residues instead of methionine. the

G-CSF used in the methods of the invention and in conjugates according to the invention may comprise a carbohydrate side chain attached to G-CSF by O-linked glycosylation at Thr 133, i.e. G-CSF is glycosylated (V. Gervais et al., Eur. J. Biochem. 1997, 247, 386-395). The structure of the carbohydrate side chain can be NeuNAc(α2-3)Gal(β1-3)[NeuNAc(α2-6)]GalNAc and (α2-3)Gal(β1-3)GalNAc (NeuNAc=N-acetylneuramine acid, GalNAc=N-acetylgalactosamine). the

It has been proposed to modify G-CSF and other polypeptides to incorporate at least one additional carbohydrate chain compared to the native polypeptide (US Patent No. 5,218,092). Depending on the host used, G-CSF expression products can be glycosylated by mammalian or other eukaryotic carbohydrates. Typically, when G-CSF is produced in eukaryotic cells, the protein is post-translationally glycosylated. As a result, carbohydrate side chains can be linked to G-CSF during biosynthesis in mammalian, especially human, insect or yeast cells. the

和 

)、来格司亭(lenograstim)( 

和 

)和那托司亭(nartograstim)( 

)的商品rhG-CSF制剂。 

和 

为非糖基化，在重组大肠杆菌细胞中产生。 

和 

为糖基化，在重组CHO细胞中产生， 为非糖基化，其在完整rhG-CSF的N-末端区有5个氨基酸置换，在重组大肠杆菌细胞中产生。  Recombinant human G-CSF (rhG-CSF) is commonly used to treat various types of leukopenia. Therefore, the available name is filgrastim (filgrastim) (

and

), Lenograstim (

and

) and nartograstim (

) commercial rhG-CSF preparation.

and

Aglycosylated and produced in recombinant E. coli cells.

and

For glycosylation, produced in recombinant CHO cells, It is aglycosylated with 5 amino acid substitutions in the N-terminal region of intact rhG-CSF, produced in recombinant E. coli cells.

作为糖基化蛋白质。根据本发明的方法和偶联物中，可使用任何非糖基化G-CSF，如 

作为非糖基化G-CSF。  Any glycosylated G-CSF can be used, such as

as glycosylated proteins. In the methods and conjugates according to the invention, any non-glycosylated G-CSF can be used, such as

As non-glycosylated G-CSF.

Furthermore, at the 1-position, G-CSF may contain a methionine amino acid residue, a serine residue or a threonine residue. the

In the context of the present invention, the term "hydroxyalkyl starch" (HAS) means a starch derivative substituted with at least one hydroxyalkyl group. A preferred hydroxyalkyl starch of the present invention has a structure according to formula (I)

Among them, the reducing end of the starch molecule is non-oxidized, and the terminal sugar unit is shown as an acetal type. According to different solvents, the acetal type may be in equilibrium with the aldehyde type. the

The term "hydroxyalkyl starch" as used in the present invention is not limited to compounds in which the terminal carbohydrate moiety comprises hydroxyalkyl groups R ₁ , R ₂ and/or R ₃ as shown for simplicity in formula (I), and also refers to compounds wherein Compounds wherein at least one hydroxyl group in any position of the carbohydrate moiety and/or the rest of the starch molecule HAS' is substituted by hydroxyalkyl _R1 , _R2 or _R3 .

Hydroxyalkyl starches may also contain two or more different hydroxyalkyl groups. the

At least one hydroxyalkyl group contained in HAS may contain two or more hydroxyl groups. According to a preferred embodiment, at least one hydroxyalkyl group comprised in HAS comprises one hydroxy group. the

The term "hydroxyalkyl starch" also includes derivatives in which the alkyl groups are mono- or polysubstituted. Herein, preference is given to alkyl being substituted by halogen, especially fluorine, or by aryl. Furthermore, the hydroxyl groups of the hydroxyalkyl groups may be esterified or etherified. the

In addition, linear or branched substituted or unsubstituted alkenyl groups can also be used instead of alkyl groups. the

Hydroxyalkyl starches are ether derivatives of starch. In addition to the ether derivatives, other starch derivatives may also be used in the present invention. For example, derivatives containing esterified hydroxyl groups may be used. These derivatives may be, for example, derivatives of unsubstituted mono- or dicarboxylic acids containing 2 to 12 carbon atoms or substituted derivatives thereof. Particularly useful are derivatives of unsubstituted monocarboxylic acids containing 2 to 6 carbon atoms, especially derivatives of acetic acid. Herein, acetyl starch, butyl starch and propyl starch are preferred. the

In addition, derivatives of unsubstituted dicarboxylic acids having 2 to 6 carbon atoms are preferred. the

For dicarboxylic acid derivatives, suitable are derivatives in which the second carboxyl group of the dicarboxylic acid is also esterified. In addition, monoalkyl ester derivatives of dicarboxylic acids are also suitable for use in the present invention. the

In the substituted mono- or dicarboxylic acid, the substituents may preferably be the same as those of the above-mentioned substituted alkyl residues. the

The technique of esterification of starch is well known in the art (see for example: Klemm D. et al., Comprehensive Cellulose Chemistry Vol. 2, 1998, Whiley-VCH, Weinheim, New York, especially Chapter 4.4, "Esterification of Cellulose (Esterification of Cellulose)” (ISBN 3-527-29489-9). 

According to a preferred embodiment of the invention, hydroxyalkyl starches according to formula (I) are used. In formula (I), the explicitly stated sugar ring and the residue denoted by HAS' together represent a preferred hydroxyalkyl starch molecule. Other sugar ring structures contained in HAS' may be the same as or different from the explicitly stated sugar ring. the

The residues R ₁ , R ₂ and R ₃ according to formula (I) are not specifically limited. According to a preferred embodiment, R ₁ , R ₂ and R ₃ are each independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 2 to 10 carbon atoms in each alkyl group. Preference is given to hydrogen and hydroxyalkyl groups having 2 to 10 carbon atoms. Hydroxyalkyl groups having 2 to 6 carbon atoms are more preferred, hydroxyalkyl groups having 2 to 4 carbon atoms are more preferred, and 2 to 4 carbon atoms are even more preferred. Therefore, "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are particularly preferred, and hydroxyethyl starch is most preferred.

Alkyl, aryl, aralkyl and/or alkaryl groups may be straight chain or branched and are suitably substituted. the

Therefore, the present invention also relates to a method as described above, wherein R ₁ , R ₂ and R ₃ are each independently hydrogen or a linear or branched hydroxyalkyl group containing 1 to 6 carbon atoms.

Thus, R ₁ , R ₂ and R ₃ may preferably be hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl, such as 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyisopropyl, hydroxy Ethyl groups, such as 2-hydroxyethyl, are particularly preferably hydrogen and 2-hydroxyethyl.

Therefore, the present invention also relates to a method and a conjugate as described above, wherein R ₁ , R ₂ and R ₃ are independently hydrogen or 2-hydroxyethyl, particularly preferably wherein R ₁ , R ₂ and R ₃ Embodiments where at least one residue is 2-hydroxyethyl.

Hydroxyethyl starch (HES) is most preferred for all embodiments of the invention. the

Accordingly, the present invention relates to a method and a conjugate as described above, wherein the polymer is hydroxyethyl starch and the polymer derivative is a hydroxyethyl starch derivative. the

Hydroxyethyl starch (HES) is a derivative of native amylopectin and can be degraded by α-amylase in vivo. HES is a substituted derivative of the carbohydrate polymer pullulan, which is present in concentrations up to 95% by weight in corn starch. HES has favorable biological properties and is clinically used as a blood volume replacement agent and for blood thinning therapy (Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; and Weidler et al., 1991, Arzneim.- Forschung/Drug Res., 41, 494-498). the

Amylopectin is composed of glucose units with α-1,4-glycosidic linkages in the main chain and α-1,6-glycosidic linkages visible at branch positions. The physicochemical properties of the molecule are mainly determined by the type of glycosidic bond. Due to the presence of the nicked α-1,4-glycosidic bonds, a helical structure of about 6 glucose monomers per turn is produced. The physicochemical and biochemical properties of polymers can be altered by substitution. Hydroxyethyl groups can be introduced by basic hydroxyethylation. Different reactivity to hydroxyethylation is possible for each hydroxyl group in the unsubstituted glucose monomer by adjusting the reaction conditions. Based on this fact, those skilled in the art can change the substitution pattern within a limited range. the

The main characteristics of HES are the molecular weight distribution and the degree of substitution. There are two possible ways of expressing the degree of substitution:

1. The degree of substitution can be expressed as the ratio of substituted glucose monomers to all glucose moieties. the

2. The degree of substitution can be expressed as a molar degree of substitution, which represents the number of hydroxyethyl groups per glucose moiety. the

In the context of the present invention, the degree of substitution expressed in DS relates to the molar degree of substitution as described above. the

HES solutions appear as polydisperse compositions in which individual molecules differ from each other in degree of polymerization, number and pattern of branching sites, and substitution pattern. Therefore, HES is a mixture of compounds of different molecular weights. Therefore, a specific HES solution is determined by the average molecular weight obtained by means of statistics. Herein, M _n is calculated as an arithmetic mean according to the number of molecules. Alternatively, the mean weight M _w (or MW) represents a unit depending on the mass of the HES.

In the context of the present invention, hydroxyethyl starch may preferably have an average molecular weight (average weight) of 1 to 300 kD. The hydroxyethyl starch may further exhibit a preferred molar degree of substitution of 0.1 to 0.8, with a preferred ratio of _C2 : _C6 substitution of hydroxyethyl ranging from 2 to 20.

The term "average molecular weight" as used in the present invention refers to the molecular weight according to Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; and Weidler et al., 1991, Arzneim.-Forschung/Drug Res., 41, 494-498 Determined weight. the

According to a preferred embodiment of the present invention, the average molecular weight of the hydroxyethyl starch used is 1 to 300 kD, more preferably 2 to 200 kD, more preferably 4 to 130 kD, more preferably 4 to 70 kD. the

是一种人造胶体，其用于例如在医疗上进行容量置换，供治疗和预防低血容量症。 

的特征为平均分子量为130,000+/-20,000D，摩尔取代程度为0.4，C2∶C6比例约9∶1。  An example of HES with an average molecular weight of about 130 kD is from the company Fresenius

is an artificial colloid used, eg, in medicine for volume replacement, for the treatment and prevention of hypovolaemia.

It is characterized by an average molecular weight of 130,000+/-20,000D, a molar substitution degree of 0.4, and a C2:C6 ratio of about 9:1.

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the hydroxyalkyl starch is hydroxyethyl starch with an average molecular weight of 4 to 70Kd. the

Preferred ranges for average molecular weight are for example: 4 to 70 kD or 10 to 70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18kD or 12 to 18kD or 4 to 12kD or 10 to 12kD or 4 to 10kD. the

According to a particularly preferred embodiment of the present invention, the average molecular weight range of hydroxyethyl starch used is above 4kD and below 70kD, for example: about 10kD, or in the range of 9 to 10kD or 10 to 11kD or 9 to 11kD, or about 12 kD, or in the range of 11 to 12 kD or 12 to 13 kD or 11 to 13 kD, or about 18 kD, or in the range of about 17 to 18 kD or 18 to 19 kD or 17 to 19 kD, or about 50 kD, or in the range of 49 to 50 kD Or in the range of 50 to 51kD or 49 to 51kD. the

With regard to the degree of substitution (DS), DS is preferably at least 0.1, more preferably at least 0.2, more preferably at least 0.4. The preferred range of DS is 0.1 to 0.8, more preferably 0.2 to 0.8, more preferably 0.3 to 0.8, even more preferably 0.4 to 0.8, still more preferably 0.1 to 0.7, more preferably 0.2 to 0.7, more preferably 0.3 to 0.7, more preferably 0.4 to 0.7. Particularly preferred DS values are for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, more preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, even more preferably 0.3, 0.4, 0.5, 0.6 , 0.7 or 0.8, still more preferably 0.4, 0.5, 0.6, 0.7 or 0.8, particularly preferably such as 0.4 and 0.7. the

Particularly preferred combinations of the molecular weight of hydroxyalkyl starches, preferably hydroxyethyl starches, and their degree of substitution DS are, for example: 10 kD and 0.4 or 10 kD and 0.7 or 12 kD and 0.4 or 12 kD and 0.7 or 18 kD and 0.4 or 18 kD and 0.7 or 50 kD and 0.4 or 50kD and 0.7. the

In another preferred embodiment of the invention, hydroxyethyl starch (used and included in the conjugates described herein) has a molecular weight of from about 20 kD to about 130 kD (i.e., about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80kD, about 90kD, about 100kD, about 110kD, about 120kD, about 130kD), preferably an average molecular weight of about 30kD to about 100kD, more preferably about 40 to about 70kD, and a degree of substitution of 0.4 to 0.8, more preferably 0.5 to 0.8. the

The term "about 30kD" herein is understood to mean that the average molecular weight is in the range of 25kD to 34kD, that is, starches having an average molecular weight of 26, 27, 28, 29, 31, 32, 33 or 34kD are also included. the

The term "about 40 kD" herein is understood to mean that the average molecular weight is in the range of 35 kD to 44 kD, ie also includes starches with an average molecular weight of 36, 37, 38, 39, 41, 42, 43 or 44 kD. the

The term "about 50 kD" herein is understood to mean that the average molecular weight is in the range of 45 kD to 54 kD, ie also includes starches with an average molecular weight of 46, 47, 48, 49, 51, 52, 53 or 54 kD. the

The term "about 60 kD" herein is understood to mean that the average molecular weight is in the range of 55 kD to 64 kD, ie also includes starches with an average molecular weight of 56, 57, 58, 59, 61, 62, 63 or 64 kD. the

The term "about 70 kD" herein is understood to mean that the average molecular weight is in the range of 65 kD to 74 kD, ie also includes starches with an average molecular weight of 66, 67, 68, 69, 71, 72, 73 or 74 kD. the

The term "about 80 kD" herein is understood to mean that the average molecular weight is in the range of 75 kD to 84 kD, ie also includes starches with an average molecular weight of 76, 77, 78, 79, 81, 82, 83 or 84 kD. the

The term "about 90 kD" herein is understood to mean that the average molecular weight is in the range of 85 kD to 94 kD, ie also includes starches with an average molecular weight of 86, 87, 88, 89, 91, 92, 93 or 94 kD. the

The term "about 100 kD" herein is understood to mean that the average molecular weight is in the range of 95 kD to 104 kD, ie also includes starches with an average molecular weight of 96, 97, 98, 99, 101, 102, 103 or 104 kD. the

The term "about 110 kD" herein is understood to mean that the average molecular weight is in the range of 105 kD to 114 kD, ie also includes starches with an average molecular weight of 106, 107, 108, 109, 111, 112, 113 or 114 kD. the

The term "about 120kD" herein is understood to mean that the average molecular weight is in the range of 115kD to 124kD, that is, starches with an average molecular weight of 116, 117, 118, 119, 121, 122, 123 or 124kD are also included. the

The term "about 130 kD" herein is understood to mean that the average molecular weight is in the range of 125 kD to 134 kD, ie also includes starches with an average molecular weight of 126, 127, 128, 129, 131, 132, 133 or 134 kD. the

The above embodiments therefore include hydroxyethyl starch (and conjugates comprising the same as described herein) with an average molecular weight of about 30 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 and methods of using the hydroxyethyl starch described herein). the

Therefore, the above embodiments also include hydroxyethyl starches with an average molecular weight of about 40 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising the hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 50 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the bifurcation compounds described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 60 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 70 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 80 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising the hydroxyethyl starch Conjugates and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 90 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the bifurcation compounds described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 100 kD, and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the dual hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 110 kD, and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the dual hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 120 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

Accordingly, the above-described embodiments also include hydroxyethyl starches having an average molecular weight of about 130 kD and a degree of substitution of 0.4 or 0.5 or 0.6 or 0.7 or 0.8, preferably 0.6, 0.7 or 0.8 (and the hydroxyethyl starches described herein comprising such hydroxyethyl starches). joints and methods of using the hydroxyethyl starch described herein). the

An example of a HES having an average molecular weight of about 130 kD is a HES having a degree of substitution of 0.2 to 0.8, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, preferably 0.4 to 0.7, such as 0.4, 0.5, 0.6 or 0.7. the

Regarding the C ₂ :C ₆ substitution ratio, the substitution ratio is preferably in the range of 2 to 20, more preferably 2 to 15, even more preferably 3 to 12.

According to another embodiment of the invention, mixtures of hydroxyethyl starches having different average molecular weights and/or different degrees of substitution and/or different _C2 : _C6 substitution ratios can also be used. Thus, mixtures of hydroxyethyl starches used may have different average molecular weights and different degrees of substitution and different _C2 : _C6 substitution ratios, or different average molecular weights and different degrees of substitution and the same or about the same _C2 : _C6 Substitution ratios, or having different average molecular weights and the same or about the same degree of substitution and different _C2 : _C6 substitution ratios, or having the same or about the same average molecular weight and different degrees of substitution and different _C2 : _C6 substitution ratios, or have different average molecular weights and the same or about the same degree of substitution and the same or about the same _C2 : _C6 substitution ratio, or have the same or about the same average molecular weight and different degrees of substitution and the same or about the same _C2 : _C6 Substitution ratio, or have the same or about the same average molecular weight and the same or about the same degree of substitution and different _C2 : _C6 substitution ratio, or have about the same average molecular weight and about the same degree of substitution and about the same _C2 : C ₆ substitution ratio.

According to different conjugates and/or different methods of the present invention, different hydroxyalkyl starches, preferably different hydroxyethyl starches and/or mixtures of different hydroxyalkyl starches, preferably different hydroxyethyl starches, can be used. the

According to one embodiment of the present invention, the functional group Z of the protein is an aldehyde group or a ketone group. Therefore, the present invention relates to a method and a conjugate as described above, wherein the functional group Z of the protein is an aldehyde group or a ketone group. the

Although the position of the aldehyde or ketone group in the protein is generally not limited, according to a preferred embodiment of the present invention, the aldehyde or ketone group is located on the carbohydrate side chain of the protein. Thus, this embodiment employs glycosylated proteins. the

Chemielexikon，Thierne Verlag Stuttgart，Germany，第9版1990，Vol.9，p.2281-2285和其中引用的文献)。此外，其也指天然碳水化合物部分(如半乳糖、N-乙酰神经氨酸和 N-乙酰半乳糖胺)等的衍生物。如果使用N-糖基化的G-CSF突变体，其碳水化合物部分可以是甘露糖。  Any glycosylated G-CSF can be used, such as as glycosylated proteins. In the context of the present invention, the term "carbohydrate side chain" refers to hydroxyaldehydes or hydroxyketones and their chemical modifications (see

Chemielexikon, Thierne Verlag Stuttgart, Germany, 9th Edition 1990, Vol. 9, p. 2281-2285 and references cited therein). Furthermore, it also refers to derivatives of natural carbohydrate moieties such as galactose, N-acetylneuraminic acid, and N-acetylgalactosamine, among others. If an N-glycosylated G-CSF mutant is used, the carbohydrate moiety can be mannose.

In an even more preferred embodiment, the aldehyde or ketone group is part of a galactose residue of the carbohydrate side chain. By removing the terminal sialic acid, this galactose residue can be reacted with the functional group A contained in the polymer or polymer derivative and then oxidized as described below. the

In another preferred embodiment, the polymer or polymer derivative comprising the functional group A is attached to the sialic acid residue of the carbohydrate side chain, preferably the terminal sialic acid residue of the carbohydrate side chain. the

Oxidation of the terminal carbohydrate moiety can be carried out chemically or enzymatically. the

Methods for chemical oxidation of carbohydrate moieties of polypeptides are well known in the art and include treatment with periodate (Chamow et al. 1992, J. Biol. Chem., 267, 15916-15922). the

By chemical oxidation it is in principle possible to oxidize any carbohydrate moiety, terminal or non-terminal. However, by choosing mild reaction conditions, it is preferably possible to oxidize the terminal sialic acid of the carbohydrate side chain to produce an aldehyde or ketone group. the

According to one embodiment of the present invention, the mild reaction conditions refer to the reaction conditions of protein and appropriate aqueous solution of periodate as follows: the preferred periodate concentration ranges from 1 to 50 mM, more preferably from 1 to 25 mM, particularly preferably from 1 to 25 mM. 10mM, such as about 1mM, the preferred reaction temperature is 0 to 40°C, particularly preferably 0 to 21°C, such as about 0°C, the preferred reaction time is 5 minutes to 5 hours, more preferably 10 minutes to 2 hours, particularly preferably 10 minutes to 1 hour , such as about 1 hour. The preferred molar ratio of periodate:protein is from 1:200 to 1:1, more preferably from 1:50 to 1:5, such as about 15:1. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the glycosylated protein is reacted with a periodate solution before the protein is reacted with the polymer or polymer derivative to generate Proteins with aldehyde or ketone groups on carbohydrate side chains. the

Alternatively, carbohydrate side chains can be enzymatically oxidized. Enzymes for oxidizing individual carbohydrate side chains are well known in the art, for example, for galactose, the enzyme is galactose oxidase. If the polypeptide has been produced in cells that allow sialic acid to be attached to carbohydrate chains, such as mammalian cells, or has been genetically modified to allow sialic acid to be attached to carbohydrate chains, if desired Oxidation of the terminal galactose moiety , then the terminal sialic acid must eventually be (partially or completely) removed. Chemical or enzymatic methods of removing sialic acid are well known in the art (Chaplin and Kennedy (eds), 1996, Carbohydrate Analysis: a practical approach, especially Chapter 5, Montreuill, Glycoproteins, p.175-177; IRL Press Practical approach series ( ISBN 0-947946-44-3)). the

According to another preferred embodiment of the present invention, the aldehyde group or ketone group can be located at the N-terminal of the protein, and can undergo appropriate oxidation reaction. Especially when an amino acid containing a hydroxyl group is located at the N-terminus of the protein, such as threonine or serine, oxidation of the N-terminal amino acid can proceed to form the ketone or aldehyde group. Threonine is the N-terminal amino acid of human G-CSF. Another N-terminal serine or threonine can be introduced into any protein showing G-CSF-like activity by molecular biological methods. The protein or a protein expressing a human amino acid sequence can be expressed in prokaryotic or eukaryotic cells, such as bacterial, mammalian, insect or yeast cells, and can be glycosylated or not. As chemical oxidation of the suitable N-terminal amino acid, oxidation with periodate is preferred by any suitable method. the

According to another preferred embodiment of the present invention, the mild reaction conditions refer to the reaction conditions of protein and suitable aqueous periodate solution as follows: the preferred periodate concentration range is 1 to 50mM, more preferably 1 to 25mM, particularly preferably 1 to 10 mM, such as about 1 mM, the preferred reaction temperature is 0 to 40 ° C, particularly preferably 0 to 21 ° C, such as about 0 ° C, the preferred reaction time is 5 minutes to 5 hours, more preferably 10 minutes to 2 hours, especially preferably 10 minutes to 1 hour, such as about 1 hour. The preferred molar ratio of periodate:protein is from 1:200 to 1:1, more preferably from 1:50 to 1:5, such as about 15:1. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the aldehyde or ketone group is located at the carbohydrate side chain of the protein and/or at the N-terminal group of the protein. the

Thus, the oligosaccharide pattern of proteins produced in eukaryotic cells has been post-translationally glycosylated, unlike proteins of human origin. Furthermore, many glycosylated proteins do not have the required number of terminal sialic acid residues to mask other carbohydrate moieties, such as galactose residues. However, these other carbohydrate moieties, such as galactose residues, may present some disadvantages if not masked, for example, the plasma half-life of the protein may be shortened in the use of the protein as a drug. It has now surprisingly been found that by providing a protein conjugate formed from a hydroxyalkyl starch polymer, preferably a hydroxyethyl starch polymer, linked by an oxime, for example as described hereinafter, directly or via at least one link Compounds, such as one or two linking compounds, covalently linked to carbohydrate moieties in carbohydrate side chains of proteins can overcome at least one of the above-mentioned disadvantages. Therefore, it is believed that by coupling a hydroxyalkyl starch polymer or a derivative thereof, preferably a hydroxyethyl starch polymer or a derivative thereof, to at least one carbohydrate side chain of a glycosylated protein, the loss of carbohydrate side chains can be compensated for. Lack of suitable terminal carbohydrate residues. According to another aspect of the present invention, there is provided a conjugated product of a hydroxyalkyl starch polymer or a derivative thereof, preferably a hydroxyethyl starch polymer or a derivative thereof, coupled with the above-mentioned oxidized carbohydrate moiety, not only The above shortcomings can be made up, and the provided protein conjugates have better properties than natural proteins in the required application fields. Therefore, each conjugate of the present invention has complementary or even synergistic effects on proteins. Even proteins that are identical to human proteins or that are themselves human proteins may not have the desired number of suitable masked terminal carbohydrate residues, such as sialic acid residues, in the native carbohydrate moiety. At this time, the provided conjugates formed by coupling hydroxyalkyl starch polymers or derivatives thereof, preferably hydroxyethyl starch polymers or derivatives thereof, with the carbohydrate moieties oxidized as described above can not only overcome and compensate The shortcomings of artificially prepared proteins can be improved, and the characteristics of natural proteins can be improved. As regards the functional groups of hydroxyalkyl starch, preferably hydroxyethyl starch or derivatives thereof, for coupling with aldehyde or ketone groups in the oxidized carbohydrate moieties of proteins, mention may be made of the functional group A disclosed hereinafter. This general concept applies not only to glycosylated G-CSF, but in principle to all glycosylated proteins lacking terminal carbohydrate residues. Among these may be mentioned erythropoietin (EPO), interferon beta 1a (IFN beta 1a), ATIII, factor VII, factor VIII, factor IX, alpha-antitrypsin (A1AT), htPA or GM-CSF. the

Accordingly, the present invention also relates to the presence of hydroxyalkyl starches, preferably hydroxyethyl starches, or derivatives thereof, on terminal carbohydrate residues, preferably sialic acid residues, which are absent from naturally or post-translationally linked carbohydrate moieties in proteins. Use, which is achieved by covalently coupling starch or a derivative thereof to at least one oxidized carbohydrate moiety in a protein having at least one ketone or aldehyde group. the

Accordingly, the present invention also relates to a method of filling the absence of terminal carbohydrate residues, preferably sialic acid residues, on naturally or post-translationally linked carbohydrate moieties in proteins by adding hydroxyalkyl starches, preferably hydroxy Ethyl starch or a derivative thereof is covalently coupled to at least one oxidized carbohydrate moiety of a protein having at least one keto or aldehyde group, preferably via an oxime linkage. the

Furthermore, the present invention also relates to a conjugate formed by covalently linking hydroxyalkyl starch, preferably hydroxyethyl starch, or a derivative thereof, to at least one oxidized carbohydrate moiety of a protein obtained from a natural source Isolated from or expressed in eukaryotic cells such as mammals, insects or yeast cells, the carbohydrate moiety has at least one ketone or aldehyde group, wherein the conjugate is used in a desired field, preferably as a drug. Among them, it has the same or better characteristics than various unmodified proteins. the

If the functional group Z of the protein is an aldehyde or ketone group, the functional group A of the polymer or its derivative contains an amino group of the formula -NH-. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the functional group A which can react with the optionally oxidized reducing end of the polymer comprises an amino group of the formula -NH-. the

According to a preferred embodiment of the present invention, the functional group A is a group with the formula R'-NH-, wherein R' is hydrogen or alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, Alkaryl or cycloalkylaryl residues, where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be directly attached to the NH group, or according to In another embodiment, the NH group can be attached via an oxygen bridge. Alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be suitably substituted. Preferred substituents may be halogen, such as F, Cl or Br. Particularly preferred residues R' are hydrogen, alkyl and alkoxy, even more preferred are hydrogen and unsubstituted alkyl and alkoxy. the

Among alkyl and alkoxy, groups having 1, 2, 3, 4, 5 or 6 carbon atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy and isopropoxy. Particular preference is given to methyl, ethyl, methoxy, ethoxy, particularly preferably methyl or methoxy. the

Accordingly, the present invention also relates to a method and conjugates as described above, wherein R' is hydrogen or methyl or methoxy. the

According to another preferred embodiment of the invention, the functional group A has the structure R'-NH-R"-, wherein R" preferably comprises the structural unit -NH- and/or the structural unit -(C=G)-, wherein G is O or S, and/or the structural unit -SO ₂ -. According to a more preferred embodiment, the functional group R" is selected from:

Wherein if G appears twice, it is O or S independently. the

Thus, preferred functional groups A containing amino- _NH2 are, for example:

wherein G is O or S, and if it occurs twice, each independently is O or S, and R' is methyl. the

A particularly preferred functional group A comprising an amino group is aminooxy

Especially preferred H ₂ NO-, and hydrazide group

Where G is preferably O. the

Accordingly, the present invention also relates to a method as described above, wherein the functional group Z of the protein is an aldehyde or ketone group and the functional group A is an aminooxy or hydrazide group. According to a particularly preferred embodiment of the invention, A is aminooxy. the

Therefore, the present invention also relates to a conjugate as described above, wherein the functional group Z of the protein is an aldehyde group or a ketone group, and the functional group A is an aminooxy group or a hydrazide group. According to a particularly preferred embodiment of the invention, A is aminooxy. the

Oxime linkages are formed when aminooxy groups of polymers or polymer derivatives react with aldehyde or ketone groups of proteins. the

Therefore, the present invention also relates to a conjugate as described above, wherein the covalent link between the protein and the polymer or polymer derivative is a functional group Z of the protein (the functional group Z is an aldehyde group or a ketone group) and a polymeric The oxime linkage formed by the reaction of the functional group A (the functional group A is aminooxy group) of the compound or polymer derivative. the

Hydrazone linkages are formed when hydrazide groups of polymers or polymer derivatives react with aldehyde or ketone groups of proteins. the

Therefore, the present invention also relates to a conjugate as described above, wherein the covalent link between the protein and the polymer or its polymer derivative is a functional group Z of the protein (the functional group Z is an aldehyde group or a ketone group) and The hydrazone formed by the reaction of the functional group A of the polymer or polymer derivative (the functional group A is a hydrazide group) is connected. the

In order to introduce the functional group A into the polymer, there are no specific limitations as long as a polymer derivative containing the functional group A is produced. the

According to a preferred embodiment of the present invention, the functional group A is introduced into the polymer by reacting the polymer with at least a bifunctional compound, one of which is reactive with at least one functional group in the polymer, at least one other functional group of which is a functional group A may alternatively be chemically modified to form functional group A. the

According to another preferred embodiment, the polymer is reacted with at least a bifunctional compound at its optionally oxidized reducing end. the

If the polymer is reacted with its non-redox end, the polymer preferably has the structure

Wherein the formula (I) includes the aldehyde form of the non-redox end. the

If the polymer is reacted with its oxidized reducing end, the polymer preferably has a structure of formula (IIa)

And/or a structure such as formula (IIb). the

The oxidation reaction of the reducing end of the polymer, preferably hydroxyethyl starch, can be carried out according to each method or combination of methods leading to the compounds having the above structure (IIa) and/or (IIb). the

Although the oxidation can be carried out according to all suitable methods or methods for producing oxidized reducing ends of hydroxyalkyl starches, it is preferably carried out using an alkaline iodine solution as described in DE 19628705 A1, the content of which document (Example A, page 9 column, lines 6 to 24) are hereby incorporated by reference. the

Each functional group that can form a chemical bond with the optionally oxidized reducing end of the hydroxyalkyl starch can be used as the functional group of the at least bifunctional compound that can react with the optionally oxidized reducing end in the polymer. the

According to a preferred embodiment of the invention, the functional group comprises the chemical structure -NH-. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein at least the functional group of the bifunctional compound, which can react with the optionally oxidized reducing end of the polymer, comprises the structure -NH-. the

According to a preferred embodiment of the present invention, the functional group of the at least bifunctional compound is a group having the structure R'-NH-, wherein R' is hydrogen or alkyl, cycloalkyl, aryl, aralkyl, aryl Cycloalkyl, alkaryl or cycloalkylaryl residues, where cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues can be directly attached to NH group, or according to another embodiment, the NH group may be linked via an oxygen bridge. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be suitably substituted. As preferred substituents mention may be made of halogens such as F, Cl or Br. Particularly preferred residues R' are hydrogen, alkyl and alkoxy, even more preferred are hydrogen and unsubstituted alkyl and alkoxy. the

Among alkyl and alkoxy groups, groups containing 1, 2, 3, 4, 5 or 6 carbon atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy and isopropoxy. Particular preference is given to methyl, ethyl, methoxy, ethoxy, particularly preferably methyl or methoxy. the

Accordingly, the present invention also relates to a method and conjugates as described above, wherein R' is hydrogen or methyl or methoxy. the

According to another preferred embodiment of the invention, the functional group of the at least bifunctional compound has the structure R'-NH-R"-, wherein R" preferably comprises the structural unit -NH- and/or the structural unit -(C=G)-, wherein G is O or S, and/or the structural unit -SO ₂ -. According to a more preferred embodiment, the functional group R" is selected from:

Wherein if G appears twice, it is O or S independently. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein at least the functional group of the bifunctional compound (which functional group can react with the optionally oxidized reducing end of the polymer) is selected from:

wherein G is O or S, and if it appears twice, it is O or S independently, and R' is methyl. the

According to an even more preferred embodiment of the invention, at least the functional group of the difunctional compound (which is reactive with the optionally oxidized reducing end of the polymer and which comprises an amino group) is aminooxy

Particularly preferred is H ₂ NO-, or a hydrazide group

Where G is preferably O. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the functional group Z of the protein is an aldehyde group or a ketone group, and the functional group of the at least bifunctional compound (which can be combined with the optionally oxidized polymer) Reducing end reaction) is aminooxy or hydrazide, preferably aminooxy. the

Accordingly, the present invention also relates to a conjugate as described above, wherein the functional group Z of the protein is an aldehyde or ketone group, and the functional group of the at least bifunctional compound (which can be combined with the optionally oxidized polymer) Reducing end reaction) is aminooxy or hydrazide, preferably aminooxy. the

According to another preferred embodiment of the invention, the at least bifunctional compound reacts with the non-oxidized reducing end of the polymer. the

According to another preferred embodiment of the invention, the at least bifunctional compound which can react with the optionally oxidized reducing end of the polymer comprises a functional group A. the

The at least bifunctional compound can react with the polymer first, and the resulting polymer derivative continues to react with the protein through the functional group A. It is also possible that the at least bifunctional compound is first reacted with the protein via the functional group A, and the resulting protein derivative is subsequently reacted with the polymer via at least one functional group contained in the residue of the at least bifunctional compound in the protein derivative. the

According to a preferred embodiment of the invention, the at least bifunctional compound is first reacted with the polymer. the

Therefore, the present invention relates to a method and a conjugate as described above, the method further comprising the step of contacting the unoxidized reducing end of the polymer with at least The bifunctional linker compound is reacted, and then the A-containing polymer derivative is reacted with the Z-containing protein. the

Any suitable spacer may be used to separate the functional group of the at least bifunctional linking compound reactive with the polymer from the functional group A of the at least bifunctional linking compound reactive with the functional group Z of the protein. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. In general, the hydrocarbon residue has at most 60 carbon atoms, preferably at most 40, more preferably at most 20, more preferably at most 10, more preferably at most 6, particularly preferably at most 4 carbon atoms. If heteroatoms are present, the spacer generally comprises 1 to 20 heteroatoms, preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2 heteroatoms. Preference is given to O as heteroatom. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl groups, or aralkyl, alkaryl, wherein the alkyl portion may be linear and/or or cyclic alkyl. According to an even more preferred embodiment of the invention, the functional groups are separated by linear hydrocarbon chains having 4 carbon atoms. According to another preferred embodiment of the invention, the functional groups are separated by linear hydrocarbon chains having 4 carbon atoms and at least one, preferably one heteroatom, particularly preferably one oxygen atom. the

According to another preferred embodiment, the at least bifunctional linking compound is a homobifunctional linking compound. Accordingly, the present invention also relates to a process for the preparation of a conjugate as described above, wherein the at least bifunctional linking compound is a homobifunctional compound. the

Therefore, with respect to the above-mentioned preferred functional groups of the linking compound, the homobifunctional linking compound preferably comprises two aminooxy groups _H2NO- or two aminooxy groups R'-O-NH- or two hydrazide groups _H2N -NH-(C=G)-, preferably aminooxy H ₂ NO- and hydrazide H ₂ N-NH-(C=O)-, particularly preferably aminooxy H ₂ NO-.

Among all possible homobifunctional compounds containing two hydrazide groups _H2N -NH-(C=O)-, hydrazides are preferred, wherein the two hydrazide groups are separated by a hydrocarbon residue having at most 60, preferably at most 40, more preferably at most 20, more preferably at most 10, more preferably at most 6, particularly preferably at most 4 carbon atoms. More preferably, the hydrocarbon residue has 1 to 4 carbon atoms, such as 1, 2, 3 or 4 carbon atoms. Most preferably, the hydrocarbon residue has 4 carbon atoms. Therefore, a homobifunctional compound of the following formula is preferred.

According to an even more preferred embodiment of the invention, the bifunctional linking compound is a carbohydrazide

As mentioned above, the present invention also relates to a method and a conjugate as described above, wherein the at least bifunctional linking compound is a homobifunctional compound and comprises two aminooxy groups. Therefore, the present invention also relates to a method and a conjugate as described above, wherein the at least bifunctional linking compound is a homobifunctional compound and comprises two aminooxy groups H ₂ NO—.

As noted above, the polymer is preferably reacted with the bifunctional linking compound at its non-oxidized reducing end prior to reaction. Thus, a preferred homobifunctional compound comprising two aminooxy groups _H2NO- is reacted with a polymer to produce a polymer derivative comprising oxime linkages.

Therefore, since the functional group Z of the protein is an aldehyde or ketone group that preferably reacts with the aminooxy group of the polymer derivative, the present invention also relates to a conjugate as described above, comprising a polymer and a protein, respectively using Oxime or cyclic aminal links covalently link linking compounds. the

Among all possible homobifunctional compounds comprising two aminooxy groups _H2NO- , preference is given to difunctional compounds in which the two aminooxy groups are separated by a hydrocarbon residue having from 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 6, particularly preferably 1 to 4 carbon atoms. More preferably, the hydrocarbon residue has 1 to 4 carbon atoms, such as 1, 2, 3 or 4 carbon atoms. Most preferably, the hydrocarbon residue has 4 carbon atoms. Even more preferably, the hydrocarbon residue has at least one heteroatom, more preferably one heteroatom, most preferably one oxygen atom. Particular preference is given to the compound O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxylamine of the formula.

Therefore, the present invention relates to a conjugate as described above, which has a structure of the following formula

and / or

HAS' is preferably HES'. Particularly preferred hydroxyethyl starches are, for example: hydroxyethyl starches with an average molecular weight of about 10 kD and a DS of about 0.4, or hydroxyethyl starches with an average molecular weight of about 10 kD and a DS of about 0.7, or hydroxyethyl starches with an average molecular weight of about 12 kD and a DS of about 0.4 Ethyl starch, or hydroxyethyl starch with an average molecular weight of about 12 kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18 kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 18 kD and a DS of about 0.7, or Hetastarch with an average molecular weight of about 50 kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 50 kD and a DS of about 0.7. the

The reaction of the non-redox end of the polymer with the linking compound is preferably carried out in an aqueous system, especially when the linking compound is a homobifunctional linking compound comprising two aminooxy groups _H2NO- .

The term "aqueous system" as used in the present invention means that the solvent or solvent mixture has a water content of at least 10% by weight, preferably at least 50% by weight, more preferably at least 80% by weight, and even more Preferably at least 90% by weight, or up to 100% by weight. The preferred reaction medium is water. the

According to another embodiment, at least one other solvent in which HAS, preferably HES, can be dissolved can be used. Examples of such solvents are eg DMF, dimethylacetamide or DMSO. the

The temperature employed during the reaction is not specifically limited so long as the reaction produces the desired polymer derivative. the

If the polymer is reacted with a homobifunctional linking compound comprising two aminooxy _H2NO- , preferably O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxylamine, the temperature is preferably from 0 to 45 °C, more preferably 4 to 30 °C, particularly preferably 15 to 25 °C.

The reaction time between the polymer and the homobifunctional linking compound containing two aminooxy H ₂ NO-, preferably O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxylamine can be adjusted according to specific needs, Usually in the range of 1 hour to 7 days, preferably in the range of 1 hour to 3 days, more preferably in the range of 2 hours to 48 hours.

The pH value of the reaction between the polymer and the homobifunctional linking compound containing two aminooxy H ₂ NO-, preferably O-[2-(2-aminooxy-ethoxy)-ethyl]hydroxylamine, can be adjusted according to specific needs , such as the chemical properties of the reactants. The pH is preferably from 4.5 to 9.5, more preferably from 4.5 to 6.5.

Specific examples of the above reaction conditions are, for example: a reaction temperature of about 25° C., and a pH of about 5.5. the

A suitable pH value of the reaction mixture can be adjusted by adding at least one suitable buffer. As preferred buffers mention may be made of sodium acetate buffer, phosphate or borate buffer. the

Once formed, the polymer derivative comprising the polymer and the bifunctional linking compound attached thereto can be isolated from the reaction mixture using at least one suitable method. If necessary, the polymer derivative can be precipitated and then isolated by at least one suitable method. the

If the polymer derivative is first precipitated, it is possible, for example at a suitable temperature, to contact the reaction mixture with at least one solvent or solvent mixture different from the solvent or solvent mixture in the reaction mixture. According to a particularly preferred embodiment of the invention, wherein an aqueous medium, preferably water, is used as solvent, the reaction mixture is contacted with 2-propanol at a temperature of preferably -20 to +50°C, particularly preferably -20 to 25°C. the

Isolation of the polymer derivatives may be carried out by a suitable method which may comprise one or more steps. According to a preferred embodiment of the invention, the polymer derivative is first isolated from the reaction mixture or the mixture of the reaction mixture with, for example, an aqueous mixture of 2-propanol, using suitable methods, such as centrifugation or filtration. In the second step, the separated polymer derivatives can be further processed, such as dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or freeze-drying and other post-treatments. According to an even more preferred embodiment of the invention, the isolated polymer derivative is first dialyzed, preferably against water, and then freeze-dried until the solvent content of the reaction product is low enough to meet the desired requirements of the product. Freeze-drying can be performed at a temperature of 20 to 35°C, preferably 20 to 30°C. the

The isolated polymer derivative is further reacted with the functional group Z of the protein through the functional group A, wherein Z is an aldehyde group or a ketone group. In the particularly preferred example where A is aminooxy _H2NO- , which can generate oxime linkages between polymer derivatives and proteins, the reaction is preferably in an aqueous medium, preferably in water, preferably at 0 to 40°C, more preferably It is preferably carried out at a temperature of 4 to 25°C, particularly preferably 15 to 25°C. The preferred pH of the reaction medium is 4 to 10, more preferably 5 to 9, particularly preferably 5 to 7. The reaction time is preferably 1 to 72 hours, more preferably 1 to 48 hours, particularly preferably 4 to 24 hours.

The conjugate can be further processed, such as dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or freeze-drying and other post-treatments. the

According to another embodiment of the invention, the functional group Z of the protein is an amino group. Therefore, the present invention relates to a method and a conjugate as described above, wherein the functional group Z of the protein is an amino group. the

According to another preferred embodiment of the invention, the functional group A reactive with the amino group as functional group Z is a reactive carboxyl group. Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the functional group Z is an amino group and the functional group A of the polymer or polymer derivative is a reactive carboxyl group. the

According to a first preferred embodiment of the invention, reactive carboxyl groups are introduced into the polymer by selective oxidation of the polymer at the reducing end. the

Therefore, the polymkeric substance that will introduce reactive carboxyl preferably has formula (IIa)

And/or a structure such as formula (IIb). the

Such as the polymer of formula (I),

Preferably, the oxidation of the reducing end of hydroxyethyl starch can be carried out according to each method or combination of methods leading to the structures (IIa) and/or (IIb) above. the

Although the oxidation can be carried out according to any suitable method or methods which can produce the oxidized reducing ends of hydroxyalkyl starches, it is preferably carried out by the alkaline iodine solution method as described in DE 19628705 A1, the content of which (Example A, page 9 column, lines 6 to 24) are incorporated herein by reference. the

All feasible methods can be used to introduce reactive carboxyl groups into polymers whose reducing ends have been selectively oxidized. the

The oxidized polymers can be used as such or as salts, such as alkali metal salts, preferably sodium and/or potassium salts. the

According to a preferred method of the invention, the polymer whose reducing end has been selectively oxidized is reacted at the oxidized reducing end with at least one alcohol, preferably with at least one acidic alcohol. Further preferred are acidic alcohols with a pK _A value of 6 to 12, more preferably 7 to 11 at 25°C. The molecular weight of the acidic alcohol is preferably 80 to 500 g/mol, more preferably 90 to 300 g/mol, particularly preferably 100 to 200 g/mol.

Suitable acidic alcohols are all HOR _A alcohols which have acidic protons and which can react with oxidized polymers to form respective reactive polymer esters, which are preferably of the formula

More preferably the following formula

Preferred alcohols are N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted phenols, such as p-nitrophenol, o, p- Dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol, such as 2,4,6 - trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. Particular preference is given to N-hydroxysuccinimides, particularly preferably N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide. All alcohols may be used alone or as an appropriate combination of two or more. In the context of the present invention, it is also possible to use compounds which release the respective alcohols, for example by addition of diesters of carbonic acid. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the polymer whose reducing end is selectively oxidized is reacted by reacting the oxidized polymer with an acidic alcohol, preferably N-hydroxysuccinimide and/or sulphonic acid. Activated by reaction with N-hydroxysuccinimide. the

According to another preferred embodiment of the present invention, the polymer whose reducing end is selectively oxidized is reacted at the oxidized reducing end with at least one carbonic acid diester R _B -O-(C=O)-OR _C , wherein R _B is combined with R _C may be the same or different. Preferably, the process produces a reactive polymer of the formula

Among them, HAS' is preferably HES'. the

Suitable carbonic acid diester compounds can be used whose alcohol components are independently the following: N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted Phenols, such as p-nitrophenol, o, p-dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5- Trichlorophenol, trifluorophenol, such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. Particular preference is given to N,N'-disuccinimidyl carbonate and sulfo-N,N'-disuccinimidyl carbonate, particularly preferably N,N'-disuccinimidyl carbonate. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the polymer selectively oxidized at the reducing end is formed by reacting the oxidized polymer with N,N'-disuccinimidyl carbonate activation. the

The acidic alcohol reacts with the polymer of oxidation or the salt of the polymer of oxidation, the molar ratio of acidic alcohol: polymer is preferably 5:1 to 50:1, more preferably 8:1 to 20:1, and the reaction temperature is preferably 2 to 40°C, more preferably 10 to 30°C, particularly preferably 15 to 25°C. The reaction time is preferably 1 to 10 hours, more preferably 2 to 5 hours, more preferably 2 to 4 hours, especially 2 to 3 hours. the

The carbonic acid diester compound is reacted with the oxidized polymer or the salt of the oxidized polymer, preferably in a diester compound:polymer molar ratio of 1:1 to 3:1, more preferably 1:1 to 1.5:1. The reaction time is preferably 0.1 to 12 hours, more preferably 0.2 to 6 hours, more preferably 0.5 to 2 hours, especially 0.75 to 1.25 hours. the

According to a preferred embodiment of the invention, the reaction of the oxidized polymer with an acidic alcohol and/or a carbonic acid diester is carried out in at least one aprotic solvent, particularly preferably at a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight in an anhydrous aprotic solvent. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. The reaction temperature is preferably 2 to 40°C, more preferably 10 to 30°C. the

At least one activator is also used in the reaction of the oxidized polymer with at least one acidic alcohol. the

Suitable activators are especially carbonyldiimidazole, carbodiimides such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), particularly preferably dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) . the

Accordingly, the present invention also relates to a process and a conjugate as described above, wherein the reducing-end oxidized polymer is reacted with an acidic alcohol in the presence of a further activating agent to produce a reactive polymeric ester. the

According to a particularly preferred embodiment of the invention, the reaction of the oxidized polymer with the carbonic acid diester and/or the acidic alcohol is carried out at a low base activity, which can be determined by adding the reaction mixture to water, wherein the water and the reaction mixture The volume ratio is 10:1. The water, substantially free of buffer, had a pH of 7 at 25°C prior to the addition. After adding the reaction mixture, the pH value is measured, and the base activity value of the reaction mixture obtained is preferably no more than 9.0, more preferably no more than 8.0, particularly preferably no more than 7.5. the

According to a preferred embodiment of the present invention, the oxidized polymer is reacted with N-hydroxysuccinimide in anhydrous DMA, in the absence of water, using EDC to selectively produce polymer N-hydroxysuccinimide of the formula Urethane

Among them, HAS' is more preferably HES'. the

Surprisingly, this reaction does not produce by-products arising from the reaction of EDC with the OH groups of HES, and unexpectedly suppresses the formation of each N-acylurea from EDC with the O-acylisourea formed from the oxidized polymer. rearrangement reaction. the

According to another preferred embodiment of the invention, the oxidized polymer is reacted with N,N'-disuccinimidyl carbonate in anhydrous DMF in the absence of an activator to selectively produce a polymer of the formula N-Hydroxysuccinimide ester

Among them, HAS' is more preferably HES'. the

The above-mentioned reactive polymer is preferably further reacted with at least one amino group of the protein to generate an amide bond. According to a preferred embodiment of the invention, the reactive polymer reacts with one amino group of the protein. the

Accordingly, the present invention relates to conjugates preferably having a structure of the formula

The N atom of the amide bond is derived from the amino group of the protein, wherein HAS' is more preferably HES', and the hydroxyethyl starch is preferably hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.4, or an average molecular weight of about 10kD and a DS of about 0.7 hydroxyethyl starch, or hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4 , or hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7. the

The reaction of the reactive polymer with the protein can be carried out by combining the reaction mixture for preparing the reactive polymer with an aqueous solution of the protein, i.e. without isolating the reactive polymer, comprising at least 10% by weight, more preferably at least 30%, still more preferably At least 50% by weight reactive polymer. Preferred aqueous protein solutions contain 0.05 to 10%, more preferably 0.5 to 5%, particularly preferably 0.5 to 2% by weight of protein, preferably have a pH of 5.0 to 9.0, more preferably 6.0 to 9.0, particularly preferably 7.5 to 8.5. the

According to the invention, it is also possible to purify the reactive polymer by at least one, preferably multiple, precipitation methods using at least one suitable precipitating agent, such as absolute ethanol, isopropanol and/or acetone, to produce a compound containing at least 10%, more preferably at least 30%, even more preferably at least 50% by weight solids of the reactive polymer. the

Purified reactive polymers can be added to aqueous protein solutions. It is also possible to add purified reactive polymer solutions to aqueous protein solutions. the

According to a preferred embodiment of the present invention, the reaction between the reactive polymer and the protein to produce amide bonds is carried out at a temperature of 2 to 40°C, more preferably 5 to 35°C, especially 10 to 30°C, and the pH is preferably 7.0 to 9.0, preferably 7.5 to 9.0, particularly preferably 7.5 to 8.5, the reaction time is preferably 0.1 to 12 hours, more preferably 0.5 to 5 hours, more preferably 0.5 to 3 hours, even more preferably 0.5 to 72 hours, particularly preferably 0.5 to 1 hour, the reactivity The molar ratio of polymer ester:protein is preferably from 1:1 to 70:1, more preferably from 5:1 to 50:1, particularly preferably from 10:1 to 50:1. the

According to another embodiment of the present invention, the reducing end-selectively oxidized polymer is reacted at its oxidized reducing end with an azolide, such as carbonyldiimidazole or carbonylbibenzimidazole, to produce a polymer with reactive carboxyl groups. In the case of carbonyldiimidazole, a reactive polymer derivative of the formula is produced

Among them, HAS' is preferably HES'. The imidazolide produced by the reaction of the polymer with the azolide can preferentially react with the amino group of the protein to form an amide bond. If there is a hydroxyl group in the protein, it can also react with the hydroxyl group to generate an ester bond, or react with the sulfur group of the protein to generate a thioester bond, or if the protein has a carboxyl group, it may also react with the carboxyl group to generate -(C=O)-O-( C=O) - connection. the

According to another embodiment of the present invention, there is a reactive carboxyl group A produced by the reaction of the selectively oxidized reducing end of the polymer with one of the above compounds, preferably with at least one acidic alcohol and/or at least one carbonic acid diester compound The polymer can be attached to the functional group Z of the protein through at least one linking compound. If a linking compound is used, the compound is at least bifunctional, having at least one functional group F1 reactive with the functional group _A of the polymer derivative, and at least one functional group F2 reactive with the functional group _Z of the protein or chemically modified Functional group F ₂ reactive with functional group Z of protein. The chemical modification can be, for example, the reaction of a functional group _F2 with a functional group _F3 of another linking compound, or oxidation or reduction of a suitable functional group _F2 . If at least one linking compound is used, the reaction is not limited to the amino groups of the protein, but depending on the chemical nature of the functional group of one or more linking compounds, it can be used with each suitable functional group of the protein such as carboxyl, reactive carboxyl, aldehyde group, keto group, thio group, amino group or hydroxyl group to form a link. If two linking compounds are used, the first linking compound used has at least one functional group F ₁ , such as amino, thio, hydroxyl or carboxyl, which can react with a reactive carboxyl group A of the polymer. Furthermore, the first linking compound has at least one other functional group _F2 which is reactive with at least one functional group _F3 of the second linking compound. The functional group _F2 may especially mention the following functional groups:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diol;

-1,2-aminoalcohols;

-1,2-Amino-thiols;

- azide;

-amino- _NH or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups, such as vinyl iodide or vinyl bromide groups or trifluoromethanesulfonate (triflate);

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is Cl, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

-group

Wherein _F is a group that can form a chemical bond with one of the above-mentioned groups, and it is preferably selected from the above-mentioned groups. Furthermore, the second linking compound has at least one functional group reactive with the functional group Z of the protein, which is eg amino, thio, carboxyl, reactive carboxyl, aldehyde, ketone or hydroxyl. If a linking compound is used to covalently link the polymer to the protein, the polymer can be reacted with the linking compound and the resulting polymer derivative reacted with the protein, or the protein can be reacted with the linking compound and the resulting protein derivative reacted with the polymer reaction. If two linking compounds L1 and L2 are used, the polymer can be reacted with L1, and the resulting polymer derivative can be reacted with L2, and the resulting polymer derivative can be reacted with protein, or the protein can be reacted with L2, and the resulting protein derivative can be reacted again. Reacted with L1, the resulting protein derivative is then reacted with a polymer. It is also possible to react polymers with L1, proteins with L2, and polymer derivatives with protein derivatives. In addition, it is also possible to react L1 and L2, the obtained compound reacts with a polymer, and the obtained polymer derivative reacts with a protein. In addition, it is also possible to react L1 and L2, the obtained compound reacts with protein, and the obtained protein derivative reacts with polymer.

According to a second preferred embodiment of the invention concerning the introduction of reactive carboxyl groups into polymers, reactive carboxyl groups are introduced into polymers whose reducing ends are not oxidized by reacting at least one hydroxyl group of the polymer with a carbonic acid diester. the

Therefore, the present invention also relates to a method and conjugates as described above, wherein A is a reactive carboxyl group, and wherein through at least one hydroxyl group of the polymer and at least one carbonic acid diester R _B —O—(C=O )-OR _C (where R _B and R _C can be the same or different) react to introduce A into the polymer that is not oxidized at the reducing end.

According to another embodiment of the present invention, at least one hydroxyl group in the unoxidized polymer at the reducing end is reacted with an azolide such as carbonyldiimidazole, carbonyl-di-(1,2,4-triazole) or carbonyldibenzimidazole to produce Polymers with reactive carboxyl groups. the

Applicable carbonic acid diester compounds can use compounds whose alcohol components are independently the following components: N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, appropriately substituted Phenols, such as p-nitrophenol, o, p-dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5- Trichlorophenol, trifluorophenol, such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. the

Especially preferred are symmetric carbonic acid diester compounds, so R _B is the same as R _C . The alcohol component of the carbonic acid diester is preferably selected from the group consisting of: N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, N-hydroxybenzotriazole and nitro- and halogen-substituted phenols. Among them, nitrophenol, dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol and pentafluorophenol are preferred, especially N, N'-disuccinimidyl carbonate and sulfo-N, N' - Disuccinimidyl carbonates, particularly preferably N,N'-disuccinimidyl carbonates.

Therefore, the present invention also relates to a hydroxyalkyl starch derivative and a process for its preparation, preferably a hydroxyethyl starch derivative, wherein at least one hydroxyl group, preferably at least two hydroxyl groups of the starch have been reacted with a carbonic acid diester compound to produce the respective Reactive esters. the

According to a preferred embodiment of the invention, the reaction of the reducing end non-oxidized polymer with at least one carbonic acid diester compound is carried out at a temperature of 2 to 40°C, more preferably 10 to 30°C, especially 15 to 25°C, preferably the reaction The time is 0.5 to 5 hours, more preferably 1 to 3 hours, particularly preferably 2 to 3 hours. the

Carbonic acid diester and/or azolide, preferred carbonic acid diester compound: The molar ratio of polymer depends on the degree of substitution of polymer, namely the relative ratio of the number of hydroxyl groups reacted with the carbonic acid diester compound and the number of hydroxyl groups in the unreacted polymer . the

According to a preferred embodiment of the present invention, the molar ratio of carbonic acid diester compound:anhydroglucose unit is 1:2 to 1:1000, more preferably 1:3 to 1:100, particularly preferably 1:10 to 1:50, so The resulting degree of substitution is from 0.5 to 0.001, preferably from 0.33 to 0.01, particularly preferably from 0.1 to 0.02. The degree of substitution was determined by UV spectroscopy. the

According to a preferred embodiment of the invention, the reaction of the reducing-end non-oxidized polymer with the carbonic acid diester is carried out in at least one aprotic solvent, particularly preferably at a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight in anhydrous aprotic solvents. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein at least one hydroxyl group in the unoxidized polymer at the reducing end reacts with a carbonic acid diester to produce a reactive ester group A in an anhydrous aprotic electrode. in a neutral solvent, preferably dimethylacetamide, dimethylformamide or a mixture thereof. the

The reaction of a reactive polymer comprising at least one reactive ester group, preferably at least two reactive ester groups, with a protein to produce at least one amide bond, preferably at least two amide bonds, can be prepared by combining a reaction mixture of reactive polymers with An aqueous solution of the protein, ie without isolating the reactive polymer, comprises at least 5% by weight, more preferably at least 10, even more preferably at least 15% by weight of reactive polymer. Preferred aqueous solutions of protein comprise 0.05 to 10%, more preferably 0.5 to 5%, particularly preferably 0.5 to 2% by weight of protein, preferably at a pH of 7.0 to 9, more preferably 7.5 to 9, particularly preferably 7.5 to 8.5. the

According to the invention, it is also possible to purify the reactive polymer by at least one, preferably multiple, precipitation using at least one suitable precipitating agent, such as absolute ethanol, isopropanol and/or acetone, to produce a compound containing at least 20%, more preferably at least 50% %, even more preferably at least 80% by weight solids of the reactive polymer. the

Purified reactive polymers can be added to aqueous protein solutions. It is also possible to add purified reactive polymer solutions to aqueous protein solutions. the

According to a preferred embodiment of the present invention, the reaction between the reactive polymer and the protein to produce at least one, preferably at least two, amide bonds is carried out at a temperature of 2 to 40°C, more preferably 5 to 35°C, particularly preferably 10 to 30°C, preferably pH 7.0 to 9.0, preferably 7.5 to 9.0, particularly preferably 7.5 to 8.5, preferably reaction time 0.5 to 5 hours, more preferably 0.5 to 3 hours, particularly preferably 0.5 to 1 hour, reactive polymer ester: protein molar ratio Preferably it is 1:1 to 70:1, more preferably 5:1 to 50:1, particularly preferably 10:1 to 50:1. the

According to a preferred embodiment of the invention, oligo- or polyprotein-substituted polymers are obtained in which protein molecules are linked to the polymer via amide bonds. the

The degree of substitution (PDS) of a protein molecule as used in the context of the present invention refers to the ratio of the glucose moieties attached to the protein relative to the total glucose moieties contained in HAS, preferably HES. the

The PDS ranges from 0.001 to 1, preferably from 0.005 to 0.5, more preferably from 0.005 to 0.2. the

According to another embodiment of the invention, polymers having reactive carboxyl groups A resulting from the reaction of at least one hydroxyl group of the polymer with one of the above compounds, preferably at least one carbonic acid diester, can be attached to proteins via at least one linking compound The functional group Z. If a linking compound is used, _the compound is at least bifunctional, having at least one functional group F1 reactive with the functional group A of the polymer derivative, and at least one functional group F2 reactive with the functional group _Z of the protein or chemically modified Functional group F ₂ reactive with functional group Z of protein. The chemical modification can be, for example, reacting a functional group _F2 with a functional group _F3 of another linking compound, or oxidizing or reducing a suitable functional group _F2 . If at least one linking compound is used, the reaction is not limited to the amino groups of the protein, but depending on the chemical nature of the functional group of one or more linking compounds, it can be used with each suitable functional group of the protein such as carboxyl, reactive carboxyl, aldehyde , keto, thio, amino or hydroxyl to form a link. If two linking compounds are used, the first linking compound used has at least one functional group F ₁ , such as amino, thio, hydroxyl or carboxyl, which can react with a reactive carboxyl group A of the polymer. Furthermore, the first linking compound has at least one other functional group _F2 which is reactive with at least one functional group _F3 of the second linking compound. The functional group _F2 may especially mention the following functional groups:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diol;

-1,2-aminoalcohols;

-1,2-Amino-thiols;

- azide;

-amino- _NH or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflate;

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is Cl, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

-group

Wherein _F3 is a group that can form a chemical bond with one of the above-mentioned groups, which is preferably selected from the above-mentioned groups. Furthermore, the second linking compound has at least one functional group reactive with the functional group Z of the protein, which is eg amino, thio, carboxyl, reactive carboxyl, aldehyde, ketone or hydroxyl. If a linking compound is used to covalently link the polymer to the protein, the polymer can be reacted with the linking compound and the resulting polymer derivative reacted with the protein, or the protein can be reacted with the linking compound and the resulting protein derivative reacted with the polymer reaction. If two linking compounds L1 and L2 are used, the polymer can be reacted with L1, and the resulting polymer derivative can be reacted with L2, and the resulting polymer derivative can be reacted with protein, or the protein can be reacted with L2, and the resulting protein derivative can be reacted again. Reacted with L1, the resulting protein derivative is then reacted with a polymer. It is also possible to react polymers with L1, proteins with L2, and polymer derivatives with protein derivatives. In addition, it is also possible to react L1 and L2, the obtained compound reacts with a polymer, and the obtained polymer derivative reacts with a protein. In addition, it is also possible to react L1 and L2, the obtained compound reacts with protein, and the obtained protein derivative reacts with polymer.

According to a particularly preferred embodiment of the present invention, the functional group Z of the protein is an amino group, and the functional group A of the polymer or its derivative is an aldehyde group, a ketone group or a hemiacetal group. According to a particularly preferred embodiment, the functional group Z is reacted with the functional group A by reductive amination. the

According to the reductive amination reaction of the present invention, wherein the polymer or polymer derivative is covalently linked to at least one amino group of the protein through at least one aldehyde group or ketone group or hemiacetal group, the reaction is preferably at 0 to 40°C, It is more preferably carried out at a temperature of 0 to 25°C, particularly preferably 4 to 21°C. The preferred reaction time is 0.5 to 72 hours, more preferably 2 to 48 hours, particularly preferably 4 to 7 hours. Aqueous media are preferred as reaction solvents. the

Accordingly, the present invention also relates to a process and a conjugate as described above, wherein the reductive amination is carried out at a temperature of 4 to 21°C. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out in an aqueous medium. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out in an aqueous medium at a temperature of 4 to 21°C. the

The term "aqueous medium" as used herein refers to a solvent or solvent mixture in which the water content is at least 10% by weight, more preferably at least 20% by weight, more preferably at least 30% by weight of the solvent involved , more preferably at least 40% by weight, more preferably at least 50% by weight, more preferably at least 60% by weight, more preferably at least 70% by weight, more preferably at least 80% by weight, even more preferably at least 90% by weight or Up to 100% by weight. The preferred reaction medium is water. the

The pH of the reaction medium is generally between 4 and 9 or between 4 and 8 or between 4 and 7.3. the

According to a preferred embodiment of the present invention, the reaction pH of the reductive amination is lower than 7.3, more preferably lower than or equal to 7, most preferably lower than 7, ie in the acidic range. Therefore, the preferred range is 3 to below 7, more preferably 3.5 to 6.5, still more preferably 4 to 6, still more preferably 4.5 to 5.5, especially preferably about 5.0, i.e. 4.6 or 4.7 or 4.8 or 4.9 or 5.0 or 5.1 or 5.2 Or 5.3 or 5.4. Preferred ranges are especially 3 to 6.9 or 3 to 6.5 or 3 to 6 or 3 to 5.5 or 3 to 5 or 3 to 4.5 or 3 to 4 or 3 to 3.5 or 3.5 to 6.9 or 3.5 to 6.5 or 3.5 to 6 or 3.5 to 5.5 Or 3.5 To 5 Or 3.5 To 4.5 Or 3.5 To 4 Or 4 To 6.9 Or 4 To 6.5 Or 4 To 6 Or 4 To 5.5 OR 4.5 to 5.5 or 4.5 to 5 or 5 to 6.9 or 5 to 6.5 or 5 to 6 or 5 to 5.5 or 5.5 to 6.9 or 5.5 to 6.5 or 5.5 to 6 or 6 to 6.9 or 6 to 6.5 or 6.5 to 6.9. the

Accordingly, the present invention also relates to a method and conjugate as described above, wherein the reductive amination is carried out at pH 7 or below, more preferably at pH 6 or below. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out at a temperature of 4 to 21° C., at a pH of 7 or below, preferably 6 or below. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out in an aqueous medium at a pH of 7 or below, preferably 6 or below. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out at a temperature of 4 to 21° C. in an aqueous medium with a pH of 7 or below, preferably 6 or below. the

The molar ratio of polymer derivative used in the reaction: protein is preferably 200:1 to 5:1, more preferably 100:1 to 10:1, particularly preferably 75:1 to 20:1. the

It has surprisingly been found that, especially in the preferred pH range mentioned above, in particular at pH below 7 and above or equal to 4, polymer derivatives can be reacted predominantly with amino groups located at the N-terminus of proteins. The term "predominantly" as used in the present invention relates to an embodiment wherein at least 80%, preferably at least 85%, of the N-terminal amino groups are available for reductive amination. It is also possible to react with at least 90% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% of the N-terminal amino groups. Although the possibility of coupling with amino groups other than the N-terminal amino group cannot be completely ruled out, it is believed that coupling by reductive amination at a pH below 7, preferably below 6 according to the present invention is substantially selected selectively at the N-terminal amino group. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the protein comprises an N-terminal amino group and at least one other amino group, the conjugate comprising a polymer predominantly coupled to the N-terminal amino group. the

According to a particularly preferred embodiment of the present invention, the present invention relates to a hydroxyalkyl starch functionalized via an aldehyde or ketone group or hemiacetal or via an aldehyde or ketone group or hemiacetal, mainly on the N-terminal amino group of the protein. A method for functionalized hydroxyalkyl starch derivatives, the method comprising reductive amination of the hydroxyalkyl starch or derivatives thereof at pH 7 or below, preferably at pH 6 or below, preferably at pH 6 or below carried out in aqueous media. the

According to the invention, aldehyde-functionalized hydroxyalkyl starches or aldehyde-functionalized hydroxyalkyl starch derivatives are preferred. the

According to another preferred embodiment, the present invention relates to a hydroxyalkyl starch functionalized with aldehyde or ketone group or hemiacetal or a hydroxyalkyl starch derivative functionalized with aldehyde or keto group or hemiacetal A method to the N-terminal amino group of a protein, the method comprising reductive amination of the hydroxyalkyl starch or derivatives thereof at pH 7 or below, preferably pH 6 or below, preferably in an aqueous medium Carried out, the hydroxyethyl starch used is preferably hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4 Hydroxyethyl starch, or hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.7, Or hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7. the

The reaction of a polymer derivative with a protein between an aldehyde or ketone or hemiacetal group and an amino group is a reductive amination to generate a Schiff's base. After the reaction, the base can be reduced with at least one reducing agent to create a stable link between the polymer derivative and the protein. It is also possible to carry out the reaction in the presence of at least one reducing agent. According to a preferred embodiment, the reductive amination reaction is carried out in the presence of at least one reducing agent. the

Preferred reducing agents are sodium borohydride, sodium cyanoborohydride, organoborane complexes such as 4-(dimethylamine)pyridine borane complex, N-ethyldiisopropylamine borane complex compound, N-ethylmorpholine borane complex, N-methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, three Ethylamine borane complex or trimethylamine borane complex. Particular preference is given to sodium cyanoborohydride. the

Accordingly, the present invention also relates to a process and conjugates as described above, wherein the reductive amination is carried out in the presence of _NaCNBH3 .

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out in an aqueous medium at pH 7 or below, preferably 6 or below, in the presence of a reducing agent, preferably NaCNBH ₃ .

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the reductive amination is carried out at a temperature of 4 to 21° C., in an aqueous medium with a pH of 7 or below, preferably 6 or below, in a reducing agent, It is preferably carried out in the presence of NaCNBH ₃ .

The molar ratio of polymer derivative:protein used in the reaction is preferably 200:1 to 10:1, more preferably 100:1 to 10:1, particularly preferably 75:1 to 20:1. the

Accordingly, the present invention also relates to a method for the preparation of a conjugate comprising reacting a polymer or polymer derivative comprising an aldehyde group with an amino group of a protein in an aqueous medium in the presence of a reducing agent, preferably is NaCNBH ₃ .

According to a first preferred embodiment of the present invention, the polymer comprises at least two aldehyde groups introduced into the polymer through an ring-opening oxidation reaction, and the polymer preferably comprises at least one structure of the following formula:

According to this embodiment of the invention, an oxidizing agent or combination of oxidizing agents capable of oxidizing at least one sugar ring of a polymer to form an open sugar ring having at least one, preferably at least two aldehyde groups may be used. The reaction is illustrated by the following reaction scheme, which shows that oxidation of the sugar ring of the polymer produces an open ring with two aldehyde groups:

Suitable oxidizing agents are in particular periodates, such as alkali metal periodates or mixtures of two or more thereof, preferably sodium and potassium periodate. the

Accordingly, the present invention also relates to a method and conjugate as described above, wherein the polymer is subjected to a ring-opening oxidation reaction using periodate to produce a polymer derivative having at least one, preferably at least two aldehyde groups . the

In this oxidation reaction, a polymer having an oxidized or non-oxidized reducing end may be used, preferably the non-oxidized type. the

Accordingly, the present invention also relates to a method and a conjugate as described above, in which a polymer having a reducing end in the non-oxidized form is used. the

The reaction temperature preferably ranges from 0 to 40°C, more preferably from 0 to 25°C, particularly preferably from 0 to 5°C. The preferred range of the reaction time is 1 minute to 5 hours, particularly preferably 10 minutes to 4 hours. The molar ratio of periodate:polymer can be suitably chosen according to the degree of oxidation desired, ie according to the number of aldehyde groups produced by the oxidation reaction. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the ring-opening oxidation reaction is carried out at a temperature of 0 to 5°C. the

The oxidation reaction of the polymer with periodate is preferably carried out in an aqueous medium, most preferably in water. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the ring-opening oxidation reaction is carried out in an aqueous medium. the

A suitable pH value of the reaction mixture can be adjusted by adding at least one suitable buffer. As preferred buffers there may be mentioned sodium acetate buffers, phosphate or borate buffers. the

The hydroxyethyl starch that carries out described ring-opening oxidation reaction is preferably the hydroxyethyl starch of average molecular weight about 10kD, DS about 0.4, or the hydroxyethyl starch of average molecular weight about 10kD, DS about 0.7, or the about 12kD of average molecular weight, DS About 0.4 hydroxyethyl starch, or hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.7 Base starch, or hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7. the

The resulting polymer derivative can be purified from the reaction mixture by at least one suitable method. If desired, the polymer derivative can be precipitated and then isolated by at least one suitable method. the

If the polymer derivative is first precipitated, it is possible, for example, to contact the reaction mixture with at least one solvent or solvent mixture different from the solvent or solvent mixture in the reaction mixture at a suitable temperature. According to a particularly preferred embodiment of the invention, wherein an aqueous medium, preferably water, is used as solvent, the reaction mixture is mixed with 2-propanol or acetone and ethanol, preferably a 1:1 mixture (v/v) (representing equal volumes of all said compounds) at a temperature of preferably -20 to +50°C, particularly preferably -20 to 25°C. the

The polymer derivatives may be isolated using a suitable method, which may comprise one or more steps. According to a preferred embodiment of the present invention, the polymer derivative is first isolated from the reaction mixture or the mixture of the reaction mixture with eg 2-propanol aqueous mixture using suitable methods such as centrifugation or filtration. In the second step, the isolated polymer derivatives can be further processed, such as after dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or lyophilization, etc. deal with. According to an even more preferred embodiment, the isolated polymer derivative is first dialyzed, preferably against water, and then lyophilized until the solvent content of the reaction product is low enough to meet the desired requirements of the product. Lyophilization can be carried out at a temperature of 20 to 35°C, preferably 20 to 30°C. the

According to a preferred embodiment, the oxidized polymer produced by the oxidation reaction is purified by at least one suitable method, such as ultrafiltration and/or dialysis, for example to remove unwanted low molecular weight salts and polymer components, thereby also providing controlled oxidative polymerization method for the molecular weight range of the compound. the

The oxidized polymer can be used directly for reaction with the protein or it can be recovered appropriately in the first step, for example by lyophilization, and then redissolved in water for conjugation to the protein in the second step. Regarding the coupling of at least one amino group of a protein with at least one aldehyde group of a polymer by reductive amination, reference can be made to the above detailed description of specific reaction parameters such as pH or temperature for the reductive amination reaction. the

According to a second preferred embodiment, the polymer is reacted with at least a bifunctional compound comprising at least one functional group M reactive with the polymer and at least one functional group M capable of reductive amination with amino groups of the protein and being an aldehyde A functional group Q of a ketone group or a hemiacetal group. the

The oxidized polymer can be used directly for reaction with the protein or it can be recovered appropriately in the first step, for example by lyophilization, and then redissolved in water for conjugation to the protein in the second step. Regarding the coupling of at least one amino group of a protein with at least one aldehyde group of a polymer by reductive amination, reference can be made to the above detailed description of specific reaction parameters such as pH or temperature for the reductive amination reaction. According to a particularly preferred embodiment of the present invention, the reductive amination is preferably carried out at a temperature of 0 to 5°C, such as about 4°C, at a pH of about 4.5 to 5.5, such as about 5.0, for a reaction time of about 20 to 30 hours, such as About 24 hours. the

According to a second preferred embodiment, the polymer is reacted with at least a bifunctional compound comprising at least one functional group M capable of reacting with the polymer and at least one functional group M capable of reductive amination with amino groups of the protein and being an aldehyde A functional group Q of a ketone group or a hemiacetal group. the

Preferably used compounds contain at least one carboxyl group or at least one reactive carboxyl group, preferably one carboxyl group or one reactive carboxyl group, in addition to an aldehyde group or a ketone group or a hemiacetal group. The aldehyde or ketone or hemiacetal group and the carboxyl or reactive carboxyl group may be separated by any suitable spacer. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. In general, the hydrocarbon residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue is an aryl residue having 5 to 7, preferably 6 carbon atoms. The hydrocarbon residue is most preferably a benzene residue. According to this preferred embodiment, the carboxyl and aldehyde groups can be located in the 1,4-position, 1,3-position or 1,2-position, preferably the 1,4-position, of the benzene ring. the

As reactive carboxyl groups, mention may be made of reactive esters, isothiocyanates or isocyanates. Preferred reactive esters are derived from N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted phenols, such as p-nitrophenol, o, p-dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol, such as 2,4 , 6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. Particular preference is given to N-hydroxysuccinimides, particularly preferably N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide. All alcohols can be used alone or in combination of two or more. Particularly preferred reactive esters are pentafluorophenyl and N-hydroxysuccinimide esters. the

Therefore, according to a preferred embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer is reacted with formylbenzoic acid. the

According to another preferred embodiment, the invention relates to a method and a conjugate as described above, wherein the polymer is reacted with pentafluorophenyl formylbenzoate. the

According to another preferred embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer is reacted with N-hydroxysuccinimide formylbenzoate. the

According to another embodiment, the invention relates to a method and a conjugate as described above, wherein the polymer is reacted with 4-(4-formyl-3,5-dimethoxyphenoxy)butanoic acid. the

The hydroxyethyl starches reacted with compounds comprising M (wherein M is preferably carboxyl or reactive carboxyl) and Q (wherein Q is aldehyde or ketone or hemiacetal) most preferably have an average molecular weight of about 10 kD, DS About 0.7 of hydroxyethyl starch. It may also be hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or an average molecular weight of about 12kD. About 18kD, DS about 0.4 hydroxyethyl starch, or average molecular weight about 18kD, DS about 0.7 hydroxyethyl starch, or average molecular weight about 50kD, DS about 0.4 hydroxyethyl starch, or average molecular weight about 50kD, DS about 0.7 0.7 hydroxyethyl starch. Particular preference is given to using hydroxyalkyl starches having reducing ends of the oxidized type, even more preferably hydroxyethyl starches. the

The resulting polymer derivatives with aldehyde or ketone or hemiacetal groups are then subjected to reductive amination with protein amino groups. Regarding the coupling of at least one amino group of the protein with at least one aldehyde group or ketone group or hemiacetal group of the polymer through reductive amination, refer to the above-mentioned specific reaction parameters such as pH or temperature for the reductive amination reaction. A detailed description. According to a particularly preferred embodiment of the invention, the reaction with the amino groups of the protein is preferably carried out at a temperature of 0 to 40°C, more preferably 0 to 25°C, particularly preferably 4 to 21°C. The preferred reaction time ranges from 30 minutes to 72 hours, more preferably from 2 to 48 hours, particularly preferably from 4 hours to 17 hours. The reaction solvent is preferably an aqueous medium. The pH of the reaction medium is preferably from 4 to 9, more preferably from 4 to 8, particularly preferably from 4.5 to 5.5. the

According to a third preferred embodiment, the optionally oxidized reducing end of the polymer is reacted with an at least bifunctional compound comprising an amino group M and a functional group Q, wherein the amino group M is reacted with the optionally oxidized reducing end of the polymer, and the functional group Q After chemical modification, aldehyde-functionalized polymer derivatives are produced, and the derivatives are reductively aminated with amino groups of proteins. the

As regards the functional group Q, the following functional groups may be mentioned in particular:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diols;

-1,2-Amino-thiols;

- azide;

-1,2-aminoalcohols;

-amino- _NH or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflate;

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is Cl, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

-group

According to a preferred embodiment of the present invention, the term "functional group Q" refers to a functional group Q comprising the chemical structure -NH-. the

According to a preferred embodiment of the invention, the functional group M is a group having the structure R'-NH-, wherein R' is hydrogen or alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl radical or cycloalkylaryl residue, wherein the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue can be attached directly to the NH group, or according to another In one embodiment, the attachment to the NH group can be through an oxygen bridge. Alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be suitably substituted. As preferred substituents mention may be made of halogens such as F, Cl or Br. Particularly preferred residues R' are hydrogen, alkyl and alkoxy, even more preferred are hydrogen and unsubstituted alkyl and alkoxy. the

Among alkyl and alkoxy groups, groups containing 1, 2, 3, 4, 5 or 6 carbon atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy and isopropoxy. Particular preference is given to methyl, ethyl, methoxy, ethoxy, particularly preferably methyl or methoxy. the

According to another embodiment of the invention, the functional group M has the structure R'-NH-R"-, wherein R" preferably comprises the structural unit -NH- and/or the structural unit -(C=G)-, wherein G is O or S , and/or the structural unit -SO ₂ -. A specific example of the functional group R" is

Wherein if G appears twice, it is O or S independently. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the functional group M is selected from:

wherein G is O or S, and if it occurs twice, each independently O or S, and R' is methyl. the

According to a particularly preferred embodiment of the invention, the functional group M is amino-NH ₂ .

The term "amino Q" refers to a functional group Q comprising the chemical structure -NH-. the

According to a preferred embodiment of the invention, the functional group Q is a group having the structure R'-NH-, wherein R' is hydrogen or alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues, wherein the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues can be attached directly to the NH group, or according to another In one embodiment, an oxygen bridge can be utilized to attach to the NH group. Alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be suitably substituted. As preferred substituents mention may be made of halogens such as F, Cl or Br. Particularly preferred residues R' are hydrogen, alkyl and alkoxy, even more preferred are hydrogen and unsubstituted alkyl and alkoxy. the

Among alkyl and alkoxy groups, groups containing 1, 2, 3, 4, 5 or 6 carbon atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy and isopropoxy. Particular preference is given to methyl, ethyl, methoxy, ethoxy, particularly preferably methyl or methoxy. the

According to another embodiment of the invention, the functional group Q has the structure R'-NH-R"-, wherein R" preferably comprises the structural unit -NH- and/or the structural unit -(C=G)-, wherein G is O or S , and/or the structural unit -SO ₂ -. According to a more preferred embodiment, the functional group R" is selected from:

Wherein if G appears twice, it is O or S independently. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the functional group Q is selected from:

Wherein G is O or S, if it appears twice, it is O or S independently, and R' is methyl. the

According to a particularly preferred embodiment of the invention, the functional group Q is amino-NH ₂ .

According to another preferred embodiment of the invention, both M and Q comprise an amino group -NH-. According to a particularly preferred embodiment, both M and Q are amino- _NH2 .

According to a preferred embodiment of the invention, the compound comprising M and Q is a homobifunctional compound, more preferably most preferably comprising amino- _NH2 , or according to other embodiments, comprising hydroxyamino-O- _NH2 or the following groups as Homobifunctional compounds with functional groups M and Q

G is preferably O. Specific examples of these compounds comprising M and Q are

or

Hydroxyl reacted with a compound comprising M (M is preferably amino-NH-, more preferably amino- _NH2 , still more preferably both M and Q comprise amino-NH-, particularly preferably both M and Q comprise amino- _NH2 ) Ethyl starch is preferably hydroxyethyl starch with an average molecular weight of about 10 kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 10 kD and a DS of about 0.7. It may also be hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4, or an average Hydroxyethyl starch with a molecular weight of about 18kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7.

If both M and Q are amino- _NH2 , M and Q may be separated by any suitable spacer. Wherein the spacer can be optionally substituted linear, branched and/or cyclic hydrocarbon residues, generally, the hydrocarbon residues have 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms. If heteroatoms are present, the spacer group generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue is an alkyl chain of 1 to 20, preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms.

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the polymer is reacted with 1,4-diaminobutane, 1,3-diaminopropane or 1,2-diaminoethane, Produce polymer derivatives. the

The reaction of the at least bifunctional compound comprising M and Q with the polymer is preferably carried out at a temperature of 0 to 100°C, more preferably 4 to 80°C, particularly preferably 20 to 80°C; the preferred reaction time ranges from 4 hours to 7 days, More preferably 10 hours to 5 days, particularly preferably 17 to 4 hours. The preferred molar ratio of at least bifunctional compound:polymer is from 10 to 200, especially from 50 to 100. the

The solvent in which the at least bifunctional compound is reacted with the polymer is preferably at least one aprotic solvent, particularly preferably anhydrous aprotic solvent with a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

It is also possible to use an aqueous medium as a solvent for the reaction of the at least bifunctional compound with the polymer. the

According to a preferred embodiment, the polymer derivative comprising a polymer and an at least bifunctional compound is chemically modified at the free functional group Q resulting in a polymer derivative comprising aldehyde or ketone or hemiacetal groups. According to this embodiment, it is preferred to react the polymer derivative with at least one at least bifunctional compound comprising a functional group reactive with the functional group Q and an aldehyde or ketone or hemiacetal group. the

Various compounds having an aldehyde or ketone group or a hemiacetal group and at least one functional group which can form a link with the functional group Q of the polymer derivative are suitable as at least bifunctional compounds. The at least one functional group is selected from the same set of functional groups as Q and is selected to be reactive with Q. In the preferred case where Q is amino- _NH2 , preference is given to using compounds which, in addition to an aldehyde or ketone or hemiacetal group, also contain at least one carboxyl group or at least one reactive carboxyl group, preferably one carboxyl group or one reactive carboxyl group. The aldehyde or ketone or hemiacetal group and the carboxyl or reactive carboxyl group may be separated by any suitable spacer. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. Generally, the hydrocarbon residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue is an aryl residue having 5 to 7, preferably 6 carbon atoms. Most preferably the hydrocarbon residue is a benzene residue. According to this preferred embodiment, the carboxyl and aldehyde groups can be located in the 1,4-position, 1,3-position or 1,2-position, preferably the 1,4-position, of the benzene ring.

Reactive esters, isothiocyanates or isocyanates may be mentioned as reactive carboxyl groups. Preferred reactive esters are derived from: N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted phenols, such as p-nitrophenol, o , p-dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol, such as 2, 4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. Particular preference is given to N-hydroxysuccinimides, particularly preferably N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide. All alcohols may be used alone or in appropriate combination of two or more. Particular preference is given to pentafluorophenyl esters and N-hydroxysuccinimide esters as reactive esters. the

Therefore, according to a preferred embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer derivative comprising Q (Q is amino- _NH2 ) is further reacted with formylbenzoic acid.

According to another embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer derivative comprising Q (Q is an amino group) is further reacted with pentafluorophenyl formylbenzoate. the

According to another embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer derivative comprising Q (Q is an amino group) is further reacted with N-hydroxysuccinimide formylbenzoate . the

According to another embodiment, the present invention relates to a method and a conjugate as described above, wherein the polymer derivative comprising Q (Q is an amino group) is further combined with 4-(4-formyl-3,5-dimethyl Oxyphenoxy) butyric acid reaction. the

For the reaction solvent of polymer derivatives comprising amino groups with, for example, formylbenzoic acid, preference is given to at least one aprotic solvent, particularly preferably anhydrous aprotic solvents with a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight . Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

The reaction is preferably carried out at a temperature of 0 to 40°C, more preferably 0 to 25°C, particularly preferably 15 to 25°C, and the reaction time is preferably 0.5 to 24 hours, particularly preferably 1 to 17 hours. the

According to a preferred embodiment, the reaction is carried out in the presence of an activating agent. Suitable activators are especially carbodiimides such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylamino Propyl)carbodiimide (EDC), particularly preferably diisopropylcarbodiimide (DIC). the

The resulting polymer derivative can be purified from the reaction mixture by at least one suitable method. If desired, the polymer derivative can be precipitated and then isolated by at least one suitable method. the

If the polymer derivative is first precipitated, it is possible, for example, to contact the reaction mixture with at least one solvent or solvent mixture different from the solvent or solvent mixture in the reaction mixture at a suitable temperature. According to a particularly preferred embodiment of the invention, wherein an aqueous medium, preferably water, is used as solvent, the reaction mixture is mixed with 2-propanol or a mixture of acetone and ethanol, preferably a 1:1 mixture (v/v) (representing an equal volume of said compound) at a temperature of preferably -20 to +50°C, particularly preferably -20 to 25°C. the

Isolation of polymer derivatives can be carried out by an appropriate method which may include one or more steps. According to a preferred embodiment of the invention, the polymer derivative is first isolated from the reaction mixture or the mixture of the reaction mixture with eg 2-propanol aqueous mixture by means of suitable methods such as centrifugation or filtration. In the second step, the separated polymer derivatives can be further processed, such as dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or lyophilization and other post-treatments . According to an even more preferred embodiment of the invention, the isolated polymer derivative is first dialyzed, preferably against water, and then lyophilized until the solvent content of the reaction product is low enough to meet the desired requirements of the product. Lyophilization can be carried out at a temperature of 20 to 35°C, preferably 20 to 30°C. the

The obtained polymer derivative containing aldehyde group or ketone group or hemiacetal group continues to carry out reductive amination reaction with amino group of protein. Regarding the coupling of at least one amino group of the protein with at least one aldehyde group or ketone group or hemiacetal group of the polymer through reductive amination, refer to the above-mentioned specific reaction parameters such as pH or temperature for the reductive amination reaction. A detailed description. According to a particularly preferred embodiment of the invention, the reductive amination is at a temperature of 0 to 10°C, such as about 1 to 8°C or 2 to 6°C, such as about 4°C, at a pH of 4.5 to 5.5, such as about 5.0 conduct. The reaction time is about 10 to 20 hours, such as 12 to 19 hours or 14 to 18 hours, such as about 17 hours or about 20 to 30 hours, such as about 24 hours. the

Therefore, according to the preferred embodiments described above, when the polymer is reacted through its oxidized reducing end, the present invention also relates to conjugates of the formula:

According to a particularly preferred embodiment, the polymer is hydroxyethyl starch, i.e. HAS' is HES', and n=2, 3 or 4, most preferably 4, as described above. Thus, when the polymer is reacted through its oxidized reducing end, the present invention also relates to conjugates of the formula

According to another preferred embodiment, the invention also relates to conjugates of the formula when the polymer is reacted via its oxidized reducing end

wherein n=2, 3 or 4, _R4 is independently hydrogen or methoxy, and when _R4 is hydrogen, m=0, when _R4 is methoxyl, m=1, HAS is preferably HES' .

In the above formulas, the nitrogen attached to the protein is derived from the amino group of the protein, and the polymer derivative is attached through the aldehyde group. the

According to the above embodiment, the functional groups M and Q comprise amino- _NH2 , where M may also be amino- _NH2 , and Q comprises β-hydroxyamino-CH(OH) _-CH2 - _NH2 , preferably β-hydroxyamino.

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the amino group Q in the compound comprising two amino groups M and Q is β-hydroxyamino-CH(OH)-CH2 _{-NH2 .}

In this case, M and Q may be separated by any suitable spacer. Wherein the spacer can be optionally substituted linear, branched and/or cyclic hydrocarbon residues. In general, the hydrocarbon residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 1 to 6, particularly preferably 1 to 2 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue has 1 to 20, preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 4 carbon atoms, particularly preferably 1 to 2 carbon atoms Alkyl chain. More preferably, M and Q are separated by a methylene group. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the polymer is reacted with 1,3-diamino-2-hydroxypropane. the

If the polymer is reacted through its oxidized reducing end, a polymer derivative of the formula is produced

Among them, HAS'=HES' is particularly preferred. the

The reaction of the at least bifunctional compound comprising M and Q, particularly preferably 1,3-diamino-2-hydroxypropane, with the polymer is preferably at a temperature of 40 to 120°C, more preferably 40 to 90°C, particularly preferably 60 to 80°C temperature. The preferred reaction time ranges from 17 to 168 hours, more preferably from 17 to 96 hours, particularly preferably from 48 to 96 hours. Preferred molar ratios of at least bifunctional compound:polymer range from 200:1 to 10:1, especially 50:1100:1. the

As the solvent for the reaction of the at least bifunctional compound with the polymer, at least one aprotic solvent is preferred, preferably an anhydrous aprotic solvent with a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

The β-hydroxyl amino group Q of the polymer derivative is generally reactive with at least a bifunctional compound comprising at least one functional group reactive with Q and further comprising at least one functional group which is an aldehyde or ketone or hemiacetal group or Functional groups that can be modified to produce aldehyde or ketone or hemiacetal groups. According to another embodiment of the present invention, the β-hydroxyamino group is directly chemically modified by chemical oxidation to generate an aldehyde group. the

The oxidation reaction can be carried out using all suitable oxidizing agents which convert the β-hydroxyamino groups into aldehyde groups. Preferred oxidizing agents are periodates, such as alkali metal periodates. Particular preference is given to sodium periodate, preferably in aqueous solution. The solution preferably has an iodate concentration of 1 to 50 mM, more preferably 1 to 25 mM, particularly preferably 1 to 10 mM. The oxidation reaction is carried out at a temperature of 0 to 40°C, preferably 0 to 25°C, particularly preferably 4 to 20°C. the

The resulting polymer derivative can be purified from the reaction mixture by at least one suitable method. If desired, the polymer derivative can be precipitated and then isolated by at least one suitable method. the

If the polymer derivative is first precipitated, it is possible, for example, to contact the reaction mixture with at least one solvent or solvent mixture different from the solvent or solvent mixture in the reaction mixture at a suitable temperature. According to a particularly preferred embodiment of the invention, wherein an aqueous medium, preferably water, is used as solvent, the reaction mixture is mixed with 2-propanol or acetone and ethanol, preferably a 1:1 mixture (v/v) (representing equal volumes of all said compounds), preferably at a temperature of -20 to +50°C, particularly preferably at a temperature of -20 to 25°C. the

Isolation of polymer derivatives can be carried out by a suitable method which may comprise one or more steps. According to a preferred embodiment of the present invention, the polymer derivative is first isolated from the reaction mixture or the mixture of the reaction mixture and, for example, 2-propanol aqueous mixture using suitable methods, such as centrifugation or filtration. In the second step, the isolated polymer derivatives can be further processed, such as after dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or lyophilization, etc. deal with. According to an even more preferred embodiment, the isolated polymer derivative is first dialyzed, preferably against water, and then lyophilized until the solvent content of the reaction product is low enough to meet the desired requirements of the product. Lyophilization can be carried out at a temperature of 20 to 35°C, preferably 20 to 30°C. the

The present invention therefore also relates to a process and conjugates as described above, wherein the oxidation of the [beta]-hydroxyamino group Q is carried out with periodate. the

Accordingly, the present invention also relates to a process for the preparation of conjugates, wherein when the polymer used has an oxidized reducing end, periodate is preferably used to oxidize polymer derivatives with β-hydroxyl amino groups, particularly preferably

And in particular HAS'=HES', resulting in polymer derivatives with aldehyde groups, particularly preferred

And in particular HAS'=HES'. the

The resulting polymer derivatives with aldehyde groups A continue to react with proteins. Therefore, the present invention also relates to a method for the preparation of conjugates, the method comprising polymer derivatives with β-hydroxyl amino groups, when the polymer used has an oxidized reducing end, the following formula is particularly preferred

And in particular HAS'=HES', reacts with amino groups of proteins. the

The resulting polymer derivatives with aldehyde groups continue to undergo reductive amination reactions with protein amino groups. For coupling of at least one amino group of a protein to at least one aldehyde group of a polymer by reductive amination, see above for details. the

Therefore, according to the above preferred embodiments, the present invention also relates to a conjugate of the following formula

In particular HAS'=HES' when the polymer used has oxidized reducing ends. In the above formula, the nitrogen attached to the protein is derived from the amino group of the protein, and the polymer derivative is attached through the aldehyde group. the

According to another embodiment of the invention, the polymer is first reacted with a suitable compound to produce a first polymer derivative comprising at least one reactive carboxyl group. The first polymer derivative is then reacted with another at least bifunctional compound, wherein at least one functional group of the other compound reacts with at least one reactive carboxyl group of the polymer derivative, and at least another A functional group is an aldehyde or ketone or hemiacetal, or a functional group that can be chemically modified to produce an aldehyde or ketone or hemiacetal, wherein the resulting aldehyde or ketone or hemiacetal contains the aldehyde or ketone or hemiacetal The polymer derivative of the group reacts with at least one amino group of the protein by reductive amination as described above. It is also possible to change the order of the mutual reactions of the individual compounds. the

According to a first alternative of this other embodiment, the process for the preparation of a polymer comprising at least one reactive carboxyl group comprises the selective oxidation of the polymer at the reducing end of the polymer, followed by making the lactone

and/or carboxylic acid

or a suitable salt of a carboxylic acid, such as an alkali metal salt, preferably sodium and/or potassium, and HAS' preferably HES', is reacted with a suitable compound to produce a polymer comprising at least one reactive carboxyl group. the

Oxidation of polymers, preferably hydroxyethyl starch, can be carried out according to individual methods or combinations of methods leading to compounds of structure (IIa) and/or (IIb) above. the

Although the oxidation reaction can be carried out according to one or more suitable methods for producing redox ends of hydroxyalkyl starches, it is preferably carried out with an alkaline iodine solution such as described in DE19628705A1, the content of this document (Example A, page 9 column, lines 6 to 24) are incorporated herein by reference. the

The introduction of reactive carboxyl groups into polymers whose reducing ends have been selectively oxidized can be carried out using all possible methods and all suitable compounds. the

According to one embodiment of the invention, the polymer whose reducing end has been selectively oxidized is reacted at its oxidized reducing end with at least one alcohol, preferably with at least one acidic alcohol, such as a pK _A value at 25° C. of 6 to 12 or 7 to 11 Acid Alcohol Reactions. The molecular weight of the acidic alcohol may range from 80 to 500 g/mol, such as 90 to 300 g/mol or 100 to 200 g/mol.

Suitable acidic alcohols are all alcohols HOR _A which have acidic protons and which can react with oxidized polymers to form respective reactive polymer esters, preferably of the formula

More preferably the following formula

Preferred alcohols are N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted phenols, such as p-nitrophenol, o, p-di Nitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol, such as 2,4,6- Trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxybenzotriazole. Particularly preferred are N-hydroxysuccinimides, particularly preferably N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide. All alcohols may be used alone or in appropriate combinations of two or more. In the context of the present invention it is also possible to use compounds which release alcohol, for example by addition of diesters of carbonic acid. the

Accordingly, the present invention also relates to a process as described above, wherein the polymer selectively oxidized at the reducing end is reacted by reacting the oxidized polymer with an acidic alcohol, preferably with N-hydroxysuccinimide and/or sulfo-N -Activated by the reaction with hydroxysuccinimide. the

According to a preferred embodiment of the present invention, the reducing end-selectively oxidized polymer is reacted at its oxidized reducing end with at least one carbonic acid diester R _B -O-(C=O)-OR _C , wherein R _B and R _C can be same or different. Preferably, the process produces a reactive polymer of the formula

Among them, HAS' is preferably HES'. the

A suitable carbonic acid diester compound can use a compound whose alcohol component is independently the following groups: N-hydroxyl succinimides, such as N-hydroxyl succinimide or sulfo-N-hydroxyl succinimide, suitable Substituted phenols such as p-nitrophenol, o,p-dinitrophenol, o,o'-dinitrophenol, trichlorophenols such as 2,4,6-trichlorophenol or 2,4,5 - trichlorophenol, trifluorophenol, such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. Particular preference is given to N,N'-disuccinimidyl carbonate and sulfo-N,N'-disuccinimidyl carbonate, particularly preferably N,N'-disuccinimidyl carbonate. the

Accordingly, the present invention also relates to a process as described above, wherein the reducing end-selectively oxidized polymer is activated by reacting the oxidized polymer with N,N'-disuccinimidyl carbonate. the

The molar ratio of the reaction of acidic alcohol with the polymer of oxidation or the salt of the polymer of oxidation is acid alcohol:polymer preferably 5:1 to 50:1, more preferably 8:1 to 20:1, preferred reaction temperature is 2 to 40°C, more preferably 10 to 30°C, particularly preferably 15 to 25°C. Preferred reaction times are 1 to 10 hours, more preferably 2 to 5 hours, more preferably 2 to 4 hours, especially 2 to 3 hours. the

The diester carbonate compound is reacted with the oxidized polymer or the salt of the oxidized polymer generally in a molar ratio of diester compound:polymer of 1:1 to 3:1, such as 1:1 to 1.5:1. The reaction time is generally 0.1 to 12 hours, such as 0.2 to 6 hours, or 0.5 to 2 hours or 0.75 to 1.25 hours. the

According to a preferred embodiment of the invention, the reaction of the oxidized polymer with an acidic alcohol and/or a carbonic acid diester is in at least one aprotic solvent, such as anhydrous a in protic solvents. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. The reaction temperature is preferably 2 to 40°C, more preferably 10 to 30°C. the

At least one other activator is used in the reaction of the oxidized polymer with at least one acidic alcohol. the

Suitable activators are especially carbonyldiimidazole, carbodiimides such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-di Methylaminopropyl)carbodiimide (EDC), particularly preferably dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC ). the

Accordingly, the present invention also relates to a process and a conjugate as described above, wherein the reducing-end oxidized polymer is reacted with an acidic alcohol, in the presence of a further activating agent, to produce a reactive polymeric ester. the

According to one embodiment of the invention, the reaction of the oxidized polymer with the carbonic acid diester and/or acidic alcohol is carried out at low base activity, which can be achieved by adding the reaction mixture to water at a water:reaction mixture volume ratio of 10:1 to make sure. Prior to the addition, the substantially buffer-free water had a pH of 7 at 25°C. After adding the reaction mixture to measure the pH value, the alkali activity value of the reaction mixture is preferably not more than 9.0, more preferably not more than 8.0, particularly preferably not more than 7.5. the

According to another embodiment of the present invention, the oxidized polymer is reacted with N-hydroxysuccinimide in anhydrous DMA, in the absence of water, using EDC to selectively produce the polymer N-hydroxysuccinimide of the formula imide ester

HAS' is more preferably HES'. the

Surprisingly, this reaction does not produce by-products arising from the reaction of EDC with the OH groups of HES, and unexpectedly suppresses the formation of each N-acylurea from EDC with the O-acylisourea formed from the oxidized polymer. rearrangement reaction. the

Therefore, according to another preferred embodiment of the present invention, the oxidized polymer is reacted with N,N'-disuccinimidyl carbonate in anhydrous DMF, in the absence of water and activator, selectively resulting in N-hydroxysuccinimide esters of the formula

Among them, HAS' is more preferably HES'. the

According to another embodiment of the present invention, the reducing end-selectively oxidized polymer is reacted at the oxidized reducing end with an azolide such as carbonyldiimidazole or carbonylbibenzimidazole to produce a polymer with reactive carboxyl groups. If carbonyldiimidazole is used, a reactive imidazolate polymer derivative of the formula is obtained

Among them, HAS' is preferably HES'. the

According to a second alternative of this other embodiment of the invention, with respect to the introduction of at least one reactive carboxyl group into the polymer, the introduction of the reactive carboxyl group into the reducing end of the polymer is by reacting at least one hydroxyl group of the polymer with a carbonic acid diester. Reactive carboxyl. the

Therefore, the present invention also relates to a method and a conjugate, wherein at least one hydroxyl group of the polymer is combined with at least one carbonic acid diester R _B -O-(C=O)-OR _C (wherein R _B and R _C can be same or different) reaction to introduce a reactive carboxyl group into the reducing end unoxidized polymer.

According to another embodiment of the invention, the reducing-end non-oxidized polymer is reacted on at least one hydroxyl group with an azolide such as carbonyldiimidazole, carbonyl-di-(1,2,4-triazole) or carbonyldibenzimidazole, Produces polymers with reactive carboxyl groups. the

Suitable carbonic diester compounds can use compounds whose alcohol components are independently the following groups: N-hydroxysuccinimides, such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably Substituted phenols such as p-nitrophenol, o,p-dinitrophenol, o,o'-dinitrophenol, trichlorophenols such as 2,4,6-trichlorophenol or 2,4,5 - trichlorophenol, trifluorophenol, such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazoles, such as hydroxybenzotriazole. the

Especially preferred are symmetric carbonic acid diester compounds, so R _B is the same as R _C . The alcohol component of the carbonic acid diester is preferably selected from: N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, N-hydroxybenzotriazole and nitro- and halogen-substituted phenols. Of these, nitrophenol, dinitrophenol, trichlorophenol, trifluorophenol, pentachlorophenol and pentafluorophenol are preferred. Particularly preferred are N,N'-disuccinimidyl carbonate and sulfo-N,N'-disuccinimidyl carbonate, particularly preferred N,N'-disuccinimidyl carbonate .

Accordingly, the present invention also relates to a hydroxyalkyl starch derivative, preferably a hydroxyethyl starch derivative, in which at least one hydroxyl group, preferably at least two hydroxyl groups, of the starch has been reacted with a carbonic acid diester compound to produce the respective reactive ester. the

According to a preferred embodiment of the present invention, the reaction of the reducing end non-oxidized polymer with at least one carbonic acid diester compound is carried out at a temperature of 2 to 40°C, more preferably 10 to 30°C, especially 15 to 25°C, preferably the reaction The time is 0.5 to 5 hours, more preferably 1 to 3 hours, particularly preferably 2 to 3 hours. the

The diester carbonate compound:polymer molar ratio depends on the degree of substitution of the polymer, ie the relative ratio of the number of hydroxyl groups reacted with the diester carbonate compound to the number of hydroxyl groups in the unreacted polymer. the

According to a preferred embodiment of the present invention, the molar ratio of carbonic acid diester compound:anhydroglucose unit is 1:2 to 1:1000, more preferably 1:3 to 1:100, particularly preferably 1:10 to 1:50, so The resulting degree of substitution is from 0.5 to 0.001, preferably from 0.33 to 0.01, particularly preferably from 0.1 to 0.02. the

According to a preferred embodiment of the invention, the reaction of the reducing-end non-oxidized polymer with the carbonic acid diester is carried out in at least one aprotic solvent, particularly preferably at a water content of not more than 0.5% by weight, preferably not more than 0.1% by weight in anhydrous aprotic solvents. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein at least one hydroxyl group of the unoxidized polymer at the reducing end reacts with a carbonic acid diester to generate a reactive carboxyl group in an anhydrous aprotic polar solvent Carried out in, the solvent is preferably dimethylacetamide, dimethylformamide or a mixture thereof. the

A reactive polymer derivative comprising at least one reactive carboxyl group, preferably as described above resulting from the reaction of a polymer with an acidic alcohol, carbonate and/or azolide, is further reacted with another at least bifunctional compound, wherein the other At least one functional group _F1 of the compound reacts with at least one reactive carboxyl group of the polymer derivative. The at least one functional group F ₁ of the other compound is not specifically limited as long as it can react with at least one reactive carboxyl group of the polymer. Preferred functional groups F ₁ are, for example: amino or hydroxyl or thio or carboxyl.

This further at least bifunctional compound comprises at least one further functional group F ₂ which is an aldehyde group or a functional group F ₂ which can be chemically modified to generate an aldehyde group. The chemical modification may be, for example, reaction of the functional group _F2 with a functional group _F3 of another linking compound, or oxidation or reduction of a suitable functional group _F2 .

If _F2 reacts with a functional group _F3 of another compound, the functional group _F2 may especially be selected from:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diol;

-1,2-aminoalcohols;

-1,2-Amino-thiols;

- azide;

-amino- _NH or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflate;

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is CI, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

;

-group

Wherein _F3 is a group that can form a chemical bond with one of the above-mentioned groups, which is preferably selected from the above-mentioned groups. In addition, the second linking compound preferably has at least one aldehyde group or ketone group or hemiacetal group that can react with the amino group of the protein by reductive amination.

the functional group _F1 and the polymer-reactive aldehyde group or ketone group or hemiacetal group in the at least bifunctional linking compound, and/or the functional groups _F1 and _F2 in the at least bifunctional linking compound reacting with the polymer, and /or the functional group _F3 and the aldehyde group or ketone group or hemiacetal group in the another at least bifunctional linking compound can be separated by any suitable spacer, respectively. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. Typically, the hydrocarbon residue has at most 60, preferably at most 40, more preferably at most 20, more preferably at most 10 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2 heteroatoms. Preference is given to O as heteroatom. The hydrocarbon residue may comprise optionally branched alkyl chains or aryl or cycloalkyl groups having, for example, 5 to 7 carbon atoms, or be aralkyl, alkaryl, wherein the alkyl portion may be linear and/or or cyclic alkyl.

Examples of compounds having functional groups _F1 and _F2 are, for example: optionally substituted diaminoalkanes having 2 to 20 carbon atoms, particularly preferably 1,2-diaminoethane, 1,3-diaminopropane and 1 , 4-Diaminobutane. Examples of preferred compounds having the functional group _F3 and an aldehyde or ketone or hemiacetal group are, for example: formylbenzoic acid, pentafluorophenyl 4-formylbenzoate, N-hydroxysuccinyl 4-formylbenzoate imidate and 4-(4-formyl-3,5-dimethoxyphenoxy)butanoic acid.

Accordingly, the present invention also relates to a process for the preparation of conjugates, comprising: reacting a polymer, preferably hydroxyethyl starch, at its optionally oxidized reducing end with a compound selected from the group consisting of acidic alcohols, dicarbonate Esters and azolides, resulting in polymer derivatives comprising at least one reactive carboxyl group, which react with at least one at least bifunctional compound, resulting in compounds comprising aldehyde or ketone or hemiacetal groups or which may be chemically modified A polymer derivative that produces a functional group of an aldehyde group or a ketone group or a hemiacetal group, optionally chemically modifying the functional group to produce a polymer derivative containing an aldehyde group or a ketone group or a hemiacetal group, and then by the aldehyde group. Reductive amination reaction of polymer derivatives with ketone group or ketone group or hemiacetal group with the amino group of protein. the

Therefore, the present invention also relates to a conjugate comprising a polymer, preferably hydroxyethyl starch, covalently linked to a protein, which is obtainable by a method for preparing the conjugate, the method comprising: the polymer Reaction at the optionally oxidized reducing end with a compound selected from the group consisting of acidic alcohols, carbonic diesters and azolides yields polymer derivatives comprising at least one reactive carboxyl group in combination with at least one at least bis Reaction of functional compounds to produce polymer derivatives comprising aldehyde groups or ketone groups or hemiacetal groups or functional groups that can be chemically modified to produce aldehyde or ketone groups or hemiacetal groups, optionally chemically modifying the functional groups to produce polymer derivatives comprising Aldehyde or ketone or hemiacetal polymer derivatives, and then reductive amination reaction between the aldehyde or ketone or hemiacetal polymer derivatives and protein amino groups. the

A specific example of a compound having a functional group _F1 and a functional group _F2 oxidized to an aldehyde group is, for example: a compound having an amino group as _F1 and a β-hydroxyamino group as _F2 . A particularly preferred example is 1,3-diamino-2-hydroxypropane. The oxidation reaction can be carried out using all suitable oxidizing agents which convert β-hydroxyamino groups into aldehyde groups. Preferred oxidizing agents are periodates, such as alkali metal periodates. Particular preference is given to sodium periodate, preferably an aqueous solution thereof is used. The solution preferably has an iodate concentration of 1 to 50 mM, more preferably 1 to 25 mM, particularly preferably 1 to 10 mM. The oxidation reaction is carried out at a temperature of 0 to 40°C, preferably 0 to 25°C, particularly preferably 4 to 20°C.

The polymer derivative produced can be purified from the reaction mixture by at least one suitable method. If desired, the polymer derivative can be precipitated and then isolated by at least one suitable method. the

If the polymer derivative is first precipitated, it is possible, for example, to contact the reaction mixture with at least one solvent or solvent mixture different from the solvent or solvent mixture in the reaction mixture at a suitable temperature. According to a particularly preferred embodiment of the invention, wherein an aqueous medium, preferably water, is used as solvent, the reaction mixture is mixed with 2-propanol or acetone and ethanol, preferably a 1:1 mixture (v/v) (representing equal volumes of all said compounds) at a temperature of preferably -20 to +50°C, particularly preferably -20 to 25°C. the

Isolation of the polymer derivatives can be carried out using a suitable method which may comprise one or more steps. According to a preferred embodiment of the present invention, the polymer derivative is first isolated from the reaction mixture or the mixture of the reaction mixture with eg 2-propanol aqueous mixture using suitable methods such as centrifugation or filtration. In the second step, the separated polymer derivatives can be further processed, such as dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or lyophilization, etc. deal with. According to an even more preferred embodiment, the isolated polymer derivative is first dialyzed, preferably against water, and then lyophilized until the solvent content of the reaction product is low enough to meet the desired requirements of the product. Lyophilization can be carried out at a temperature of 20 to 35°C, preferably 20 to 30°C. the

According to another preferred embodiment of the present invention, the functional group Z of the protein reacting with the functional group A of the polymer or polymer derivative is a thiol group. the

中，Cys 18例如存在于 

中。  The thiol group may itself be present in the protein. In addition, it is also possible to introduce thiol groups into proteins according to an appropriate method. Among them chemical methods may be mentioned. If there is a disulfide bond in the protein, the -SS- structure can be reduced to obtain a thiol group. It is also possible to convert the amino group in the polypeptide into an SH group by reacting the polypeptide with a compound having at least two different functional groups through the amino group, one of which can react with the amino group, and the other is a SH group or a precursor of the SH group , such as N-succinimidyl-S-acetylthioacetate, N-succinimidyl-S-acetylthiopropionate or N-succinimidyl-3-(pyridine Dithio) propionate. It is also possible to introduce an SH group by mutation of a protein, e.g. introducing another cysteine into a protein, exchanging an amino acid for a cysteine, or e.g. removing a cysteine from a protein so that another cysteine in the protein Cystine cannot form disulfide bonds. Most preferably, the polymer is linked to a free cysteine of the protein, particularly preferably Cys 17 or Cys 18, where Cys 17 is present for example in

In, Cys 18 exists for example in

middle.

According to a first embodiment, the functional group Z of the protein is a thiol group, the functional group A of the polymer is a haloacetyl group, and wherein the introduction method of A comprises: a polymer having at least two functional groups and Each at least difunctional compound containing an amino group is reacted to produce a polymer derivative having at least one functional group containing an amino group, which is then reacted with a monohalogen-substituted acetic acid and/or a reactive monohalogen-substituted acetic acid derivative. the

For at least bifunctional compounds having at least two functional groups each containing an amino group, there may be all compounds which can react with the optionally oxidized reducing end of the polymer to produce a polymer derivative comprising The reactive amino groups of acetic acid and/or reactive monohalogen substituted acetic acid derivatives. the

According to a preferred embodiment, one functional group of the at least bifunctional compound (which functional group reacts with the optionally oxidized reducing end of the polymer) is selected from:

Wherein G is O or S, and if it appears twice, it is independently O or S, and R' is methyl. the

According to a particularly preferred embodiment of the invention, the functional group of the at least bifunctional compound which reacts with the optionally oxidized reducing end is amino- _NH2 . According to another preferred embodiment, the functional groups, most preferably amino groups, react with the oxidized reducing ends of the polymer.

According to a preferred embodiment of the invention, the functional group of the at least bifunctional compound which reacts with monohalogen-substituted acetic acid and/or reactive monohalogen-substituted acetic acid derivatives is amino- _NH2 .

The functional groups of the at least bifunctional compound are preferably both amino-NH ₂ (this functional group is combined with the optionally oxidized reducing end of the polymer, preferably the oxidized reducing end, and monohalogen substituted acetic acid and/or reactive monohalogen substituted acetic acid derivative reaction), may be separated by any suitable spacer. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. Suitable substituents are in particular alkyl, aryl, aralkyl, alkaryl, halogen, carbonyl, acyl, carboxyl, carboxyl ester, hydroxyl, thio, alkoxy and/or alkylthio. In general, the hydrocarbon residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue is an alkyl chain having 1 to 20, preferably 2 to 10, particularly preferably 2 to 8 carbon atoms. Therefore, preferred at least difunctional compounds are difunctional amino compounds, particularly preferably 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane alkanes, 1,4-diaminobutane, 1,3-diaminopropane and 1,2-diaminoethane.

According to another preferred embodiment, the at least bifunctional compound is diaminopolyethylene glycol, preferably diaminopolyethylene glycol of the formula

H ₂ N-(CH ₂ -CH ₂ -O) _m -CH ₂ -CH ₂ -NH ₂

Where m is an integer, m is preferably 1, 2, 3 or 4. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the polymer is mixed with 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1 , 5-diaminopentane, 1,4-diaminobutane, 1,3-diaminopropane and 1,2-diaminoethane react at their oxidized reducing ends to produce polymer derivatives of the following formula

Where n=2, 3, 4, 5, 6, 7 or 8, the polymer is particularly preferably HES. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the polymer is reduced to _H2N- ( _CH2 -CH2 _- O) _m - _CH2 - _CH2 - _NH2 in its oxidation terminal reaction, wherein m is 1, 2, 3 or 4, resulting in a polymer derivative of the formula

where m=1, 2, 3 or 4, and the polymer is particularly preferably HES. the

The oxidation of the reducing end of the polymer, preferably hydroxyethyl starch, can be carried out according to various methods or combinations of methods which lead to compounds of structure (IIa) and/or (IIb). the

Although the oxidation can be carried out according to one or more suitable methods which all lead to the oxidized reducing ends of hydroxyalkyl starches, it is preferably carried out using alkaline iodine solutions, as described, for example, in DE 19628705 A1, the content of which (Example A, column 9 , lines 6 to 24) are hereby incorporated by reference. the

The polymer derivative produced by reacting the polymer with the at least bifunctional compound is then reacted with monohalogen substituted acetic acid and/or reactive monohalogen substituted acetic acid derivatives. the

As monohalogen-substituted acetic acid or reactive acid, preference is given to Cl-substituted, Br-substituted and I-substituted acetic acids, particularly preferably acetyl chloride. the

If the halogen-substituted acid itself is used, it is preferred to react the acid with the polymer derivative in the presence of an activator. Suitable activators are especially carbodiimides such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylamino Propyl)carbodiimide (EDC), particularly preferably dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the polymer, preferably HES, is mixed with a diamino compound, preferably a diaminoalkane with 2 to 8 carbon atoms or H ₂ N-(CH ₂ -CH ₂ -O) _m -CH ₂ -CH ₂ -NH ₂ (wherein m=1, 2, 3 or 4) reacts, and the obtained polymer derivative is then reacted with Br-substituted and I-substituted acetic acid in an activator, preferably EDC reaction in the presence of .

Therefore, the present invention also relates to a polymer derivative of the formula

Wherein X=Cl, Br or I, n=2, 3, 4, 5, 6, 7 or 8, the polymer is particularly preferably HES, or a polymer derivative of the following formula

Where X = Cl, Br or I, m = 1, 2, 3 or 4, the polymer is particularly preferably HES. the

The reaction of the polymer derivative with the halogen-substituted acetic acid is preferably in an aqueous system, preferably in water, at a pH of preferably 3.5 to 5.5, more preferably 4.0 to 5.0, especially preferably 4.5 to 5.0, at preferably 4 to 30° C., more preferably It is preferably carried out at a reaction temperature of 15 to 25°C, particularly preferably 20 to 25°C, and the reaction time is preferably 1 to 8 hours, more preferably 2 to 6 hours, particularly preferably 3 to 5 hours. the

A reaction mixture comprising a polymer derivative including a polymer, an at least bifunctional compound and a halogen-substituted acetic acid can be used to react with the protein itself. According to a preferred embodiment of the present invention, the polymer derivative is isolated from the reaction mixture, preferably using ultrafiltration followed by precipitation, optionally washing and vacuum drying. the

The reaction of polymer derivatives with proteins is preferably carried out in an aqueous system. the

The reaction of the polymer derivative and the protein is preferably at pH 6.5 to 8.5, more preferably 7.0 to 8.5, particularly preferably 7.5 to 8.5; at preferably 4 to 30°C, more preferably 15 to 25°C, particularly preferably 20 to 25°C The reaction is carried out at the reaction temperature; the reaction time is preferably 0.5 to 8 hours, more preferably 1 to 6 hours, particularly preferably 2 to 5 hours. the

The reaction of the polymer derivative with the thiol group of the protein produces a thioether bond between the polymer derivative and the protein. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the polymer, preferably HES, is mixed with a diamino compound, preferably a diaminoalkane with 2 to 8 carbon atoms or H ₂ N—(CH ₂ -CH ₂ -O) _m -CH ₂ -CH ₂ -NH ₂ (where m = 1, 2, 3 or 4) reacts, and the resulting polymer derivative is then reacted with Br-substituted and I-substituted acetic acid in the presence of the activator In the presence, preferably in the presence of EDC, the resulting polymer derivative reacts with the thiol groups of the protein to produce a conjugate comprising a thioether linkage between the protein and the polymer derivative.

Therefore, the present invention also relates to a conjugate of the following formula

Where n=2, 3, 4, 5, 6, 7 or 8, the polymer is particularly preferably HES, or a conjugate of the following formula

Where m=1, 2, 3 or 4, the polymer is particularly preferably HES. The hydroxyethyl starch is preferably a hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4 , or hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.7, or an average molecular weight of about 0.7 50kD, hydroxyethyl starch with a DS of about 0.4, or hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7. the

According to a second embodiment, the functional group Z of the protein is a thiol group and the functional group A of the polymer comprises a maleimide group. the

According to this embodiment, there are several possibilities to generate conjugates. Typically, the polymer is reacted at its optionally oxidized reducing end with at least one at least bifunctional compound comprising a functional group reactive with the optionally oxidized reducing end of the polymer, and at least one comprising A maleimide group or a functional group chemically modified to produce a polymer derivative comprising a maleimide group. According to a preferred embodiment, this functional group is chemically modified to produce a polymer derivative comprising maleimide groups. the

Accordingly, the present invention relates to a method and conjugates as described above, produced by reacting a polymer derivative comprising maleimide groups with a thiol group of a protein, the method comprising optionally oxidizing the polymer in its The reducing end of is reacted with an at least bifunctional compound comprising a functional group U that can react with the optionally oxidized reducing end, the at least bifunctional compound further comprising a functional group W that can be chemically modified to produce a maleimide group, the method The method also includes chemically modifying the functional group W to generate a maleimide group. the

The functional group U may be any functional group reactive with an optionally oxidized reducing end of the polymer. the

According to a preferred embodiment of the invention, the functional group U comprises the chemical structure -NH-. the

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the functional group U comprises the structure -NH-. the

According to a preferred embodiment of the invention, the functional group U is a group having the structure R'-NH-, wherein R' is hydrogen or alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl radical or cycloalkylaryl residue, wherein the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residue can be attached directly to the NH group, or according to another In one embodiment, the attachment to the NH group can be through an oxygen bridge. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues may be suitably substituted. Preferred substituents may be halogen such as F, Cl or Br. Particularly preferred residues R' are hydrogen, alkyl and alkoxy, even more preferred are hydrogen and unsubstituted alkyl and alkoxy. the

Among alkyl and alkoxy groups, groups containing 1, 2, 3, 4, 5 or 6 carbon atoms are preferred. More preferred are methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy and isopropoxy. Particular preference is given to methyl, ethyl, methoxy, ethoxy, particularly preferably methyl or methoxy. the

Accordingly, the present invention also relates to a method and conjugates as described above, wherein R' is hydrogen or methyl or methoxy. the

According to another preferred embodiment of the invention, the functional group U has the structure R'-NH-R"-, wherein R" preferably comprises the structural unit -NH- and/or the structural unit -(C=G)-, wherein G is O or S, and/or the structural unit -SO ₂ -.

According to a more preferred embodiment, the functional group R" is selected from:

Wherein if G appears twice, it is independently O or S respectively. the

Therefore, the present invention also relates to a method and a conjugate as described above, wherein the functional group U is selected from:

Wherein G is O or S, and if it occurs twice, it is independently O or S, and R' is methyl. the

According to a more preferred embodiment of the invention, U comprises amino- _NH2 .

According to one embodiment of the invention, the at least bifunctional compound is chemically modified by reacting a polymer derivative comprising W with another at least bifunctional compound comprising a functional group reactive with W and further comprising a maleimide group. Functional group W. the

As the functional group W and the functional group reactive with W in the other at least bifunctional compound, the following functional groups may be mentioned in particular:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diol;

-1,2-aminoalcohols;

-1,2-amino-thiols;

- azide;

-amino- _NH or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflate;

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is Cl, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

-group

Wherein W and the functional group of the other at least bifunctional compound are respectively groups capable of forming a chemical bond with the above-mentioned one group. the

According to a still more preferred embodiment of the present invention, W comprises amino-NH ₂ .

According to a preferred embodiment of the present invention, both W and the further functional group are groups from the above list of groups. the

According to one embodiment of the invention, one of these functional groups is a thio group. In this particular example, the further functional group is preferably selected from:

wherein Hal is Cl, Br or I, preferably Br or I. the

According to a particularly preferred embodiment of the invention, one of these functional groups is selected from reactive esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide, or have the structural unit O-N, where N is a heteroaryl part of the compound, or for example an aryloxy compound with a substituted aryl residue such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl, or a carboxyl group optionally converted into a reactive ester. In this particular example, the other functional group comprises the chemical structure -NH-. the

According to a particularly preferred embodiment of the invention, W comprises the structure -NH-, the further at least bifunctional compound comprising a reactive ester and a maleimide group. the

See the functional groups above for functional groups W comprising the structure -NH-, where W may be the same as U or different. According to a preferred embodiment of the present invention, U is the same as W. More preferably, both U and W contain amino groups. Particularly preferably, both U and W are amino-NH ₂ .

According to one embodiment of the invention, the polymer can be reacted with an at least bifunctional compound comprising U and W at its non-oxidized reducing end in an aqueous medium. According to a preferred embodiment, in which U and W are both amino groups, the reaction employs a polymer having an oxidized reducing end, in at least one aprotic solvent, particularly preferably at a water content of not more than 0.5% by weight, preferably not more than 0.1% carried out in an anhydrous aprotic solvent with a weight ratio. Suitable solvents are in particular dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. the

Especially when U and W are both amino-NH ₂ , U and W may be separated by any suitable spacer. Wherein the spacer may be an optionally substituted linear, branched and/or cyclic hydrocarbon residue. Suitable substituents are alkyl, aryl, aralkyl, alkaryl, halogen, carbonyl, acyl, carboxyl, carboxyl ester, hydroxyl, thio, alkoxy and/or alkylthio. In general, the hydrocarbon residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms. If heteroatoms are present, the spacer generally contains 1 to 20, preferably 1 to 8, particularly preferably 1 to 4 heteroatoms. The hydrocarbon residue may comprise optionally branched alkyl chains having, for example, 5 to 7 carbon atoms or aryl or cycloalkyl, or aralkyl, alkaryl, wherein the alkyl moiety may be linear and /or cyclic alkyl. According to an even more preferred embodiment, the hydrocarbon residue is an alkyl chain having 1 to 20, preferably 2 to 10, more preferably 2 to 6, particularly preferably 2 to 4 carbon atoms.

Accordingly, the present invention also relates to a method and a conjugate as described above, wherein the polymer is combined at its oxidized reducing end with 1,4-diaminobutane, 1,3-diaminopropane or 1,2-diaminobutane Aminoethane reacts to produce polymer derivatives of the following formula

Where n=2, 3 or 4, the polymer is preferably HES. the

According to the preferred embodiment described above, the polymer derivative comprising amino groups is then reacted with an at least bifunctional compound comprising reactive ester groups and maleimide groups. The reactive ester and maleimide groups can be separated by a suitable spacer. For the spacer, see the spacer between the functional groups U and W. According to a preferred embodiment of the present invention, there are 1 to 10, preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 4, more preferably 1 between the reactive ester group and the maleimide group. to 2, particularly preferably 1, hydrocarbon chain separation of carbon atoms. According to another preferred embodiment, the reactive ester is a succinimide ester, and according to a particularly preferred embodiment, the at least bifunctional compound comprising a maleimide group and a reactive ester group is N-(α-maleimide to imidoacetoxy) succinimide ester. the

Therefore, the present invention also relates to a polymer derivative of the formula

Where n=2, 3 or 4, the polymer is preferably HES. the

The polymer derivatives containing maleimide groups are further reacted with thiol groups of the protein to produce conjugates containing polymer derivatives linked to the protein via thioether groups. the

Accordingly, the present invention also relates to conjugates comprising proteins and polymers of the formula

Wherein n=2, 3 or 4, preferably 4, the polymer is preferably HES, wherein the S atom in the formula is derived from Cys 17 or Cys 18 of the protein. The hydroxyethyl starch is preferably a hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 10kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.4 , or hydroxyethyl starch with an average molecular weight of about 12kD and a DS of about 0.7, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.4, or a hydroxyethyl starch with an average molecular weight of about 18kD and a DS of about 0.7, or an average molecular weight of about 0.7 50kD, hydroxyethyl starch with a DS of about 0.4, or hydroxyethyl starch with an average molecular weight of about 50kD and a DS of about 0.7. the

The reaction of the polymer derivative comprising maleimide group and the thiol group of protein is preferably carried out in a buffered aqueous system, preferably at a pH of 5.5 to 8.5, more preferably 6 to 8, particularly preferably 6.5 to 7.5, preferably the reaction The temperature is 0 to 40°C, more preferably 0 to 25°C, particularly preferably 4 to 21°C, and the reaction time is preferably 0.5 to 24 hours, more preferably 1 to 20 hours, particularly preferably 2 to 17 hours. A suitable pH value of the reaction mixture can be adjusted by adding at least one suitable buffer. Preferred buffers may be mentioned sodium acetate buffers, phosphate or borate buffers, containing urea preferably in a concentration of 0 to 8M, more preferably 2 to 8M, particularly preferably 4 to 8M, and/or containing a preferred concentration of It is 0 to 1% (w/v), more preferably 0.4 to 1% (w/v), particularly preferably 0.8 to 1% (w/v) of SDS. the

The conjugate can be further processed, such as dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reverse chromatography, HPLC, MPLC, gel filtration and/or lyophilization and other post-treatments. the

Therefore, the present invention also relates to a conjugate obtainable by the above method. the

Therefore, the present invention also relates to a conjugate obtainable by the above method, wherein A is a reactive carboxyl group, wherein by reacting at least one hydroxyl group of the polymer with a carbonic acid diester, introducing A, and wherein the conjugate comprises one polymer molecule and at least one, especially 1 to 10, protein molecules linked to the polymer via amide bonds. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony stimulating factor (G-CSF), and it has the following properties formula structure

and / or

wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl,

Wherein G is selected from O and S, preferably O, and

wherein L is an optionally suitably substituted linear, branched and/or cyclic hydrocarbon residue optionally containing at least one heteroatom, preferably an alkyl, aryl, aralkyl, heteroatom having 2 to 60 carbon atoms Aryl, heteroaralkyl residues. the

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction, which does not contain the carbon atoms of the carbohydrate moiety that are part of the oxime link in the N=C double bond. the

The present invention also relates to a conjugate as described above, wherein -L- is -(CH ₂ )n-, wherein n=2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2 , 3, 4, 5, 6, more preferably 2, 3, 4, especially preferably 4.

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony stimulating factor (G-CSF), and it has the following properties Formula structure:

and / or

wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

Wherein G is selected from O and S, preferably O. the

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction, which does not contain the carbon atoms of the carbohydrate moiety that are part of the oxime link in the N=C double bond. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony stimulating factor (G-CSF), and it has the following properties Formula structure:

and / or

wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein L is an optionally suitably substituted linear, branched and/or cyclic hydrocarbon residue which optionally contains at least one heteroatom, preferably an alkyl, aryl, aralkyl, heteroatom having 2 to 60 carbon atoms Aryl, heteroaralkyl residues. the

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction, which does not contain the carbon atoms of the carbohydrate moiety that are part of the oxime link in the N=C double bond. the

The above two structures illustrate a structure in which the cross-linking compound is attached to the reducing end of HAS via an oxime linkage, and in which the terminal sugar unit of HES is of the open type, and a structure of the type with each cyclic aminal, The cross-linking compound is connected to the reducing end of HES through the oxygen amino group, and the terminal sugar unit of HES is cyclic. These two structures can simultaneously exist in a state of mutual equilibrium. the

The present invention also relates to a conjugate as described above, wherein -L- is

-[(CR _a R _b ) _m G] _n [CR _c R _d ] _o -

wherein R _a , R _b , R _c , and R _d are independently hydrogen, alkyl, aryl, preferably hydrogen, wherein G is selected from O and S, preferably O, and wherein

m is 1, 2, 3 or 4, wherein the residues R _a and R _b in m CR _a R _b groups may be the same or different;

n is 0 to 20, preferably 0 to 10, more preferably 1, 2, 3, 4, 5, most preferably 1 or 2;

o is 0 to 20, preferably 0 to 10, more preferably 1, 2, 3, 4, 5, most preferably 1 or 2, wherein the residues R _c and R _d in o CR _c R _d groups can be the same or different.

The present invention also relates to a conjugate as described above, wherein R _a , R _b , R _c , and R _d are hydrogen, m=2, n=1, o=2.

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), and the protein is granulocyte colony-stimulating factor (G-CSF), which has the following properties formula structure

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl.

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction which does not contain the nitrogen atom of the amino group which is part of the amide bond. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has the following properties Formula structure:

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein the linkage -O-(C=O)- is formed by the reaction of a carboxyl or reactive carboxyl group with a hydroxyl group of the HAS molecule. the

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction which does not contain the nitrogen atom of the amino group which is part of the amide bond. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has the following properties Formula structure:

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein L is an optionally substituted linear, branched and/or cyclic hydrocarbon residue, optionally containing at least one heteroatom, having 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 2 carbon atoms, particularly preferably 1 carbon atom, L is especially CH ₂ .

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction which does not contain the nitrogen atom of the amino group which is part of the aminomethyl linkage. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has the following properties Formula structure:

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein L _and L are _each independently optionally substituted linear, branched and/or cyclic hydrocarbon residues, which optionally contain at least one heteroatom, comprising alkyl, aryl, aralkylheteroalkyl and / or a heteroaralkyl moiety, the residue has 1 to 60, preferably 1 to 40, more preferably 1 to 20, more preferably 1 to 10 carbon atoms, and

Wherein D is a link, preferably a covalent bond formed by a suitable functional group _F2 connecting _L1 and a suitable functional group _F3 connecting _L2 .

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction which does not contain the nitrogen atom of the amino group which is part of the aminomethyl linkage. the

The present invention also relates to a conjugate as described above, wherein L ₁ is -(CH ₂ )n-, wherein n=2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2, 3, 4, 5, 6, more preferably 2, 3, 4, especially preferably 4.

The present invention also relates to a conjugate as described above, wherein L ₂ comprises an optionally suitably substituted aryl moiety, preferably an aryl moiety comprising 6 carbon atoms, L ₂ is particularly preferably C ₆ H ₄ , or wherein L ₂ is -(CH ₂ )n-, wherein n=2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2, 3, 4, 5, 6, more preferably 2, 3, 4.

The present invention also relates to a conjugate as described above, wherein it is selected from:

-C-C-double bond or C-C-triple bond or aromatic C-C-bond;

- thiol or hydroxyl;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

-1,2-diol;

-1,2-Amino-thiols;

- azide;

-1,2-aminoalcohols;

-amino-NH ₂ or amino derivatives comprising the structural unit -NH-, such as aminoalkyl, aminoaryl, aminoaralkyl or alkarylamino;

- hydroxyamino-O-NH ₂ , or derivatives of hydroxyamino containing the structural unit -O-NH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyaralkylamino or hydroxyalkarylamino;

- alkoxyamino, aryloxyamino, aralkoxyamino or alkaryloxyamino, each comprising the structural unit -NH-O-;

-residues having a carbonyl group-Q-C(=G)-M, wherein G is O or S, and M is for example:

---OH or -SH;

--Alkyloxy, aryloxy, aralkyloxy or alkaryloxy;

--Alkylthio, arylthio, aralkylthio or alkarylthio;

--Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy; 

- activated esters, such as esters of hydroxylamine, which have an imine structure such as N-hydroxysuccinimide or have the structural unit O-N, wherein N is part of a heteroaryl compound, or wherein G = O and Q absent, such as aryloxy compounds with substituted aryl residues such as pentafluorophenyl, p-nitrophenyl or trichlorophenyl;

Wherein Q does not exist or is NH or a heteroatom, such as S or O;

--NH- _NH2 or -NH-NH-;

_--NO2 ;

- nitrile;

- carbonyl, such as aldehyde or ketone;

- carboxyl;

--N=C=O group or -N=C=S group; 

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflate;

--C≡C-H;

--(C=NH ₂ Cl)-O alkyl

--(C=O) _-CH2 -Hal group, wherein Hal is Cl, Br or I;

--CH=CH- _SO2- ;

- a disulfide group comprising the structure -S-S-;

-group

-group

And wherein _F3 is a functional group that can form a chemical bond with _F2 , preferably selected from the above-mentioned groups, _F2 preferably contains a part -NH-, more preferably contains an amino group, _F3 preferably contains a part -(C=G)-, more preferably Preferably -(C=O)-, more preferably a part of -(C=G)-G-, even more preferably -(C=O)-G-, particularly preferably -(C=O)-O, D is particularly preferably amide bond.

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has the following properties Formula structure:

Part of the carbon atoms of -CH ₂ -N ₂ - are derived from the aldehyde groups introduced into the polymer through the ring-opening oxidation reaction, where HAS" refers to those that do not contain the aldehyde groups produced by the ring-opening oxidation reaction and react with the amino groups in the protein Hydroxyalkyl starch molecule in which the nitrogen atom is derived from the amino group of the protein.

The abbreviation "protein" used in the above formula refers to the G-CSF molecule used in the reaction which does not contain the nitrogen atom of the amino group which is part of the amide bond. the

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has The following structure:

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein L is an optionally substituted linear, branched and/or cyclic hydrocarbon residue optionally containing at least one heteroatom comprising alkyl, aryl, aralkylheteroalkyl and/or heteroaralkyl moiety, the residue has 2 to 60, preferably 2 to 40, more preferably 2 to 20, more preferably 2 to 10 carbon atoms, and

The sulfur atom is derived from the cysteine residue or disulfide group of the protein. the

The present invention also relates to a conjugate as described above, wherein -L- is

-[(CR _a R _b ) _m G] _n [CR _c R _d ] _o -

wherein R _a , R _b , R _c , and R _d are independently hydrogen, alkyl, aryl, preferably hydrogen, wherein G is selected from O and S, preferably O, and wherein

m is 1, 2, 3 or 4, most preferably 2, wherein the residues R _a and R _b in m groups CR _a R _b may be the same or different;

n is 1 to 20, preferably 1 to 10, most preferably 1, 2, 3 or 4;

o is 1 to 20, preferably 1 to 10, more preferably 1, 2, 3, 4, 5, more preferably 1 or 2, most preferably 1, wherein the residues R _c and R in the o groups CR _c R _{d d} may be the same or different;

or

in

n is 0; and

o is 2 to 20, preferably 2 to 10, more preferably 2, 3, 4, 5, 6, 7 or 8, wherein the residues R _c and R _d in o groups CR _c R _d may be the same or different.

The present invention also relates to a conjugate comprising a protein and a polymer or a derivative thereof, wherein the polymer is hydroxyalkyl starch (HAS), the protein is granulocyte colony-stimulating factor (G-CSF), and it has the following properties Formula structure:

Wherein R ₁ , R ₂ and R ₃ are independently hydrogen or hydroxyalkyl, hydroxyaryl, hydroxyaralkyl or hydroxyalkaryl having 1 to 10 carbon atoms, preferably hydrogen or hydroxyalkyl, more preferably hydrogen or hydroxyethyl, and

wherein L is an optionally substituted linear, branched and/or cyclic hydrocarbon residue optionally containing at least one heteroatom comprising alkyl, aryl, aralkylheteroalkyl and/or heteroaralkyl moiety, the residue has 2 to 60, preferably 2 to 40, more preferably 2 to 20, more preferably 2 to 10 carbon atoms, and

The sulfur atom is derived from the cysteine residue or disulfide group of the protein. the

The present invention also relates to a conjugate as described above, wherein -L- is

-[(CR _a R _b ) _m G] _n [CR _c R _d ] _o -

wherein R _a , R _b , R _c , and R _d are independently hydrogen, alkyl, aryl, preferably hydrogen, wherein G is selected from O and S, preferably O, and wherein

m is 1, 2, 3 or 4, most preferably 2, wherein the residues R _a and R _b in m groups CR _a R _b may be the same or different;

n is 1 to 20, preferably 1 to 10, most preferably 1, 2, 3 or 4;

o is 1 to 20, preferably 1 to 10, more preferably 1, 2, 3, 4, 5, more preferably 1 or 2, most preferably 1, wherein the residues R _c and R in the o groups CR _c R _{d d} may be the same or different;

or

in

n is 0; and

o is 2 to 20, preferably 2 to 10, more preferably 2, 3, 4, 5, 6, 7 or 8, wherein the residues R _c and R _d in o groups CR _c R _d may be the same or different.

The present invention also relates to any conjugate as described above, wherein the hydroxyalkyl starch is hydroxyethyl starch. the

The present invention also relates to any conjugate as described above, wherein the molecular weight of the hydroxyethyl starch is 2 to 200 kD, preferably 4 to 130 kD, more preferably 4 to 70 kD. the

According to another aspect, the present invention relates to a conjugate as described above, or a conjugate obtainable by the above method, for use in a method of treatment of the human or animal body. the

In addition, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned conjugate or a conjugate that can be prepared by the above-mentioned method. the

The term "therapeutically effective amount" used in the present invention means an amount that can provide a therapeutic effect for a specific condition and administration regimen. Administration is preferably by route of administration. The particular route is chosen according to the condition being treated. Administration is preferably carried out as part of a formulation comprising a suitable carrier such as a polysorbate, a suitable diluent such as water and/or a suitable adjuvant such as sorbitol. The required dosage will depend on the severity of the condition being treated, the individual response of the patient, the method of administration employed, and the like. the

Therefore, in a preferred embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable diluent, adjuvant and/or carrier, particularly preferably for G-CSF therapy. the

The pharmaceutical composition is preferably used in the treatment of pathologies characterized by decreased hematopoietic or immune function or related diseases. the

Therefore, the present invention also relates to the use of a pharmaceutical composition as described above comprising the above-mentioned conjugate or a conjugate obtainable by the above-mentioned method for the preparation of a medicament for the treatment of a disease characterized by decreased hematopoietic or immune function. the

According to a preferred embodiment, the disease characterized by reduced hematopoietic or immune function is the result of chemotherapy, radiotherapy, infectious disease, severe chronic neutropenia or leukemia. Therefore, the present invention also relates to the use of a pharmaceutical composition as described above comprising the above-mentioned conjugate or a conjugate obtainable by the above-mentioned method in the preparation of a medicament for the treatment of diseases characterized by decreased hematopoietic or immune function, wherein The disease is the result of chemotherapy, radiation therapy, infectious disease, severe chronic neutropenia, or leukemia. the

Individuals treated with the pharmaceutical compositions of the present invention are preferably administered intravenously or subcutaneously. For this purpose, the pharmaceutical compositions can be administered as sterile solutions. the

The present invention is further illustrated by the following figures, tables and examples, which do not limit the scope of the present invention in any way. the

Description of drawings

Figure 1a

的SDS page分析。使用XCell Sure Lock Mini Cell(Invitrogen GmbH，Karlsruhe，D)和Consort E143电源(CONSORTnv，Tumhout，B)进行凝胶电泳。根据制造商的说明，使用12％Bis-Tris凝胶和还原条件下的MOPS SDS电泳缓冲液(均来自InvitrogenGmbH，Karlsruhe，D)。  Figure 1a shows the HES-G-CSF conjugate prepared according to Example 2.1(a)

SDS page analysis. Gel electrophoresis was performed using an XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions.

Plus2(Invitrogen GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: G-CSF ( ) crude product after coupling with HES as described in Example 2.1(a).

Lane C: G-CSF starting material. the

Figure 1b

Figure 1b shows the HES-G-CSF conjugate prepared according to Example 2.1(a) SDS page analysis. Gel electrophoresis was performed using XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions.

Lane A: protein marker Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

)根据实施例2.1(a)所述与HES偶联后的粗产物。  Lane B: G-CSF (

) Crude product after coupling with HES as described in Example 2.1(a).

Lane C: G-CSF starting material. the

figure 2

Figure 2 shows the SDSpage analysis of the HES-G-CSF conjugate prepared according to Example 2.1(b), G-CSF from Strathmann Biotec AG, Hamburg, D. Gel electrophoresis was performed using an XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. the

Plus2(Invitrogen  GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: Crude product after coupling of G-CSF and HES 10/0.4 in 0.1M NaOAc buffer pH 5.0. the

Lane C: crude product after coupling of G-CSF and HES 10/0.7 in 0.1M NaOAc buffer pH 5.0. the

Lane D: crude product after coupling of G-CSF and HES 50/0.4 in 0.1M NaOAc buffer pH 5.0. the

Lane E: crude product after coupling of G-CSF and HES 50/0.7 in 0.1M NaOAc buffer pH 5.0. the

Lane F: G-CSF starting material. the

image 3

Figure 3 shows the SDSpage analysis of the HES-G-CSF conjugate prepared according to Example 2.2, G-CSF from Strathmann Biotec AG, Hamburg, D. Gel electrophoresis was performed using XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. the

Plus2(Invitrogen  GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、 3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: crude product after coupling of G-CSF and oxidized HES 10/0.7 in 0.1 M NaOAc buffer pH 5.0. the

Lane C: crude product after coupling of G-CSF and oxidized HES 50/0.4 in 0.1 M NaOAc buffer pH 5.0. the

Lane D: crude product after coupling of G-CSF and oxidized HES 50/0.7 in 0.1 M NaOAc buffer pH 5.0. the

Lane E: G-CSF starting material. the

Figure 4

或 

采用XCell Sure LockMini Cell(Invitrogen GmbH，Karlsruhe，D)和Consort E143电源(CONSORTnv，Tumhout，B)进行凝胶电泳。根据制造商的说明，使用12％Bis-Tris凝胶和还原条件下的MOPS SDS电泳缓冲液(均来自Invitrogen GmbH，Karlsruhe，D)。  Figure 4 shows the SDS page analysis of the HES-G-CSF conjugate prepared according to Example 2.3, the G-CSF is

or

Gel electrophoresis was performed using XCell Sure LockMini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions.

Plus2(Invitrogen GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: crude product (i-N) according to example 2.3. the

Lane C: crude product (ii-N) according to example 2.3. the

Lane D: crude product (iii-N) according to example 2.3. the

Lane E: crude product (iv-N) according to example 2.3. the

Lane F: crude product (i-G) according to example 2.3. the

Lane G: crude product (ii-G) according to example 2.3. the

Lane H: crude product (iii-G) according to example 2.3. the

Lane I: crude product (iv-G) according to example 2.3. the

Lane J:

Figure 5

Figure 5 shows the SDSpage analysis of the HES-G-CSF conjugate prepared according to Example 2.4, G-CSF from Strathmann Biotec AG, Hamburg, D. Gel electrophoresis was performed using XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. the

Plus2(Invitrogen GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: crude product (vi) according to example 2.4. the

Lane C: crude product (v) according to example 2.4. the

Lane D: G-CSF starting material. the

Lane E: protein markers Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane F: crude product (ix) according to example 2.4. the

Lane G: crude product (viii) according to example 2.4. the

Lane H: crude product (vii) according to example 2.4. the

Lane I: G-CSF starting material. the

Figure 6

Figure 6 shows the SDSpage analysis of the HES-G-CSF conjugate prepared according to Example 2.5, G-CSF from Strathmann Biotec AG, Hamburg, D. Gel electrophoresis was performed using XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 10% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. the

Plus2(Invitrogen  GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: crude product according to example 2.5. the

Lane C: G-CSF starting material. the

Figure 7

或 采用XCell Sure LockMini Cell(Invitrogen GmbH，Karlsruhe，D)和Consort E143电源(CONSORTnv，Tumhout，B)进行凝胶电泳。根据制造商的说明，使用12％Bis-Tris凝胶和还原条件下的MOPS SDS电泳缓冲液(均来自Invitrogen GmbH，Karlsruhe，D)。  Figure 7 shows the SDS page analysis of the HES-G-CSF conjugate prepared according to Example 3, the G-CSF is

or Gel electrophoresis was performed using XCell Sure LockMini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions.

Plus2(Invitrogen  GmbH，Karlsruhe，D)。分子量标记物由上至下为：188kD、98kD、62kD、49kD、38kD、28kD、17kD、14kD、6kD、3kD。  Lane A: protein marker

Plus2 (Invitrogen GmbH, Karlsruhe, D). The molecular weight markers from top to bottom are: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD.

Lane B: crude product (x) according to example 3. the

Lane C: crude product (xi) according to example 3. the

Lane D: crude product (xii) according to example 3. the

Lane E: crude product (xiii) according to example 3. the

Lane F: crude product according to example 3 (xiv). the

Lane G: crude product (xv) according to example 3. the

Figure 8

Figure 8 shows the in vitro results of Example 6. the

In the figure, the x-axis represents the concentration in pg/ml, and the y-axis represents the number of cells/100,000. In this figure, the following abbreviations refer to:

G-CSF/A32 G-CSF conjugate prepared according to Example 2.5

G-CSF/A33 G-CSF initial material for the conjugate of Example 2.5

G-CSF/A57 Unmodified

G-CSF/A58 Unmodified

G-CSF/A60 G-CSF conjugate prepared according to Example 4.2

Figure 9

Figure 9 shows the HPGPC chromatogram of the crude coupling reaction product according to Example 4.1. The following parameters were used in the HPGPC analysis:

Column: Superose 12 HR 10/30 300×10mm I.D.(Pharmacia)

Eluent: 27.38 mM Na ₂ HPO ₄ ; 12.62 mM NaH ₂ PO ₄ ; 0.2 M NaCl; 0,005% NaN ₃ in 1 liter demineralized water

Flow rate: 0.24ml/hour

Detector 1: MALLS detector

Detector 2: UV(280nm)

Detector 3: RI (Refractive Index Detector)

A represents the result of detector 1 and B represents the result of detector 2. the

Figure 10

Figure 10 shows the HPGPC chromatogram of the coupling reaction product according to Example 4.1, wherein the reaction by-products such as the mixture of unreacted oxo-HES and free N-hydroxysuccinimide and the solvent content are all adopted 10Kd ultrafiltration membrane Downregulated by cryogenic centrifugation. HPGPC analysis employs the following parameters:

Column: Superose 12HR 10/30 300×10mm I.D.(Pharmacia)

Eluent: 27.38 mM Na ₂ HPO ₄ ; 12.62 mM NaH ₂ PO ₄ ; 0.2 M NaCl; 0.005% NaN ₃ in 1 L demineralized water

Flow rate: 0.24ml/hour

Detector 1: MALLS detector

Detector 2: UV(280nm)

Detector 3: RI (Refractive Index Detector)

A represents the result of detector 1 and B represents the result of detector 2. the

Figure 11

HPGPC analysis employs the following parameters:

Column: Superose 12 HR 10/30 300×10mm I.D.(Pharmacia)

Eluent: 27.38 mM Na ₂ HPO ₄ ; 12.62 mM NaH ₂ PO ₄ ; 0.2 M NaCl; 0.005% NaN ₃ in 1 L demineralized water

Flow rate: 0.24ml/hour

Detector 1: MALLS detector

Detector 2: UV(280nm)

Detector 3: RI (Refractive Index Detector)

A represents the result of detector 1 and B represents the result of detector 2. the

Figure 12

HPGPC analysis employs the following parameters:

Column: Superose 12 HR 10/30 300×10mm I.D.(Pharmacia)

Eluent: 27.38 mM Na ₂ HPO ₄ ; 12.62 mM NaH ₂ PO ₄ ; 0.2 M NaCl; 0.005% NaN ₃ in 1 L demineralized water

Flow rate: 0.24ml/hour

Detector 1: MALLS detector

Detector 2: UV(280nm)

Detector 3: RI (Refractive Index Detector)

A represents the result of detector 1 and B represents the result of detector 2. the

Figure 13

Figure 13 shows the SDS-PAGE analysis of flow-through and eluate of HES-modified G-CSF (A32) after chromatography on DEAE-Sepharose CL-6B. 1.5% of the indicated fractions were desalted by ultrafiltration, dried in a SpeedVac, and added to a 12.5% polyacrylamide gel. the

Figure 14

Figure 14 shows the MALDI/TOF spectrum of the G-CSF starting material (sample A33). the

Figure 15

Figure 15 shows the MALDI/TOF profile of HES-modified G-CSF (sample A32). the

Figure 16

Figure 16 shows the MALDI/TOF profile of HES-modified G-CSF (sample A60). the

Figure 17

Figure 17 shows the gel electrophoresis of the reaction mixture of Example 7.2(b). Gel electrophoresis was performed using an XCellSure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a ConsortE143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. Gels were stained using Roti-Blue (Carl Roth GmbH+Co. KG, Karlsruhe, D) according to the manufacturer's instructions. the

Lane A: protein marker Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D), molecular weight markers from top to bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD. the

Lane B: the crude product after coupling hG-CSF with the HES derivative prepared in Example 7.1(d). the

Lane C: the crude product after coupling hG-CSF with the HES derivative prepared in Example 7.1(b). the

Lane D: the crude product after coupling hG-CSF with the HES derivative prepared in Example 7.1(j). the

Lane E: reaction control: HES 50/07. the

Figure 18

Figure 18 shows the gel electrophoresis of the reaction mixture of Example 7.2(d). Gel electrophoresis was performed using an XCellSure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a ConsortE143 power supply (CONSORTnv, Tumhout, B). A 12% Bis-Tris gel and MOPS SDS running buffer under reducing conditions (both from Invitrogen GmbH, Karlsruhe, D) were used according to the manufacturer's instructions. Gels were stained using Roti-Blue (Carl Roth GmbH+Co. KG, Karlsruhe, D) according to the manufacturer's instructions. the

Lane A: protein marker Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D), the molecular weight markers from top to bottom are: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD. the

Lane B: hG-CSF after buffer exchange as described in Example 7.2(c). the

Lane C: crude product after coupling of hG-CSF to a HES derivative prepared as described in Example 7.1(f). the

Lane D: crude product after coupling of hG-CSF to a HES derivative prepared as described in Example 7.1(h). the

Figure 19

Figure 19 shows the HPGPC chromatogram of the product of the coupling reaction according to Example 7.3 (MALLS detector: upper panel; UV detector: lower panel). HPGPC analysis employs the following parameters:

Column: Superose 12 HR 10/30 300×10mm I.D.(Pharmacia)

Eluent: 27.38 mM Na ₂ HPO ₄ ; 12.62 mM NaH ₂ PO ₄ ; 0.2M NaCl; 0.005% NaN ₃ in 1 L demineralized water

Flow rate: 0.24ml/hour

Detector 1: MALLS detector

Detector 2: UV(280nm)

Detector 3: RI (Refractive Index Detector)

Figure 20

Figure 20 shows the results of the mitogenicity assay of Example 7.4. Y-axis represents NFS-60-cell number/ml, X-axis represents concentration in pg/ml. the

Figure 21

Figure 21 shows the results of the in vivo assay of Example 7.5. the

Example

Example 1 : Synthesis of aldehyde-functionalized hydroxyethyl starch

Example 1.1(a) : Synthesis of selective oxidation of the reducing end of hydroxyethyl starch by periodate oxidation and incubation at 0°C

Take 100mg of oxo-HES10/0.4 (MW=10kD, DS=0.4, prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1), dissolve in 5ml of 20mM sodium phosphate buffer pH 7.2, and cool to 0°C . 21.4 mg of sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was dissolved in 5 ml of the same buffer and cooled to 0°C. The two solutions were mixed and after incubation at 0°C for 10 minutes, 0.73 ml of glycerol was added and the reaction mixture was incubated at 21°C for 10 minutes. The reaction mixture was dialyzed against water for 24 hours (SnakeSkin dialysis tubing, cut-off value 3.5 Kd, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. the

Example 1.1(b) : Synthesis of selective oxidation of the reducing end of hydroxyethyl starch by periodate oxidation and incubation at 21°C

Take 100mg of oxo-HES10/0.4 (MW=10kD, DS=0.4, prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1), dissolved in 5ml of 20mM sodium phosphate buffer pH 7.2. 21.4 mg of sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was dissolved in 5 ml of the same buffer. The two solutions were mixed and after incubation at 21°C for 10 minutes, 0.73 ml of glycerol was added and the reaction mixture was incubated at 21°C for 10 minutes. The reaction mixture was dialyzed against water for 24 hours (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. the

Example 1.2(a): Synthesis of aldehyde-functionalized hydroxyethyl starch by oxidizing the unredox end of hydroxyethyl starch with periodate and incubating at 0°C

Take 100mg HES10/0.4 (MW=10kD, DS=0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D) dissolved in 5ml 20mM sodium phosphate buffer pH 7.2, cooled to 0°C. 21.4 mg of sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was dissolved in 5 ml of the same buffer and cooled to 0°C. The two solutions were mixed and after incubation at 0°C for 10 minutes, 0.73 ml of glycerol was added and the reaction mixture was incubated at 21°C for 10 minutes. The reaction mixture was dialyzed against water for 24 hours (SnakeSkin dialysis tubing, cut-off value 3.5 kD, PerbioSciences Deutschland GmbH, Bonn, D) and lyophilized. the

Example 1.2(b) : Using periodate to oxidize the non-redox end of hydroxyethyl starch and incubate at 21°C to synthesize aldehyde-functionalized hydroxyethyl starch

Take 100mg of HES10/0.4 (MW=10kD, DS=0.4, manufactured by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D) dissolved in 5ml of 20mM sodium phosphate buffer pH 7.2. 21.4 mg of sodium periodate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was dissolved in 5 ml of the same buffer. The two solutions were mixed and after incubation at 21°C for 10 minutes, 0.73 ml of glycerol was added and the reaction mixture was incubated at 21°C for 10 minutes. The reaction mixture was dialyzed against water for 24 hours (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. the

Example 1.3: Synthesis of aldehyde-functionalized hydroxyethyl starch from amino-functionalized hydroxyethyl starch and formylbenzoic acid

Oxo-HES10/0.4 (MW=10kD, DS=0.4) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. the

Take 5.1 g (0.51 mmol) of oxo-HES10/0.4 dissolved in 15 ml of anhydrous dimethyl sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D), and add dropwise to 5.1 ml (51 mmol) under nitrogen A solution of 1,4-diaminobutane in 10 ml of anhydrous dimethyl sulfoxide was stirred at 40°C for 19 hours. The reaction mixture was added to a mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was centrifuged, washed with a mixture of 20ml ethanol and 20ml acetone, and redissolved in 80ml water. The solution was dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) and then lyophilized. 67% (3.4 g) of amino-HES10/0.4 were obtained. the

Take 150mg of 4-formylbenzoic acid and 230mg of 1-hydroxyl-1H-benzotriazole (both from Sigma-Aldrich Chemie GmbH, Taufkirchen, D) and dissolve in 10ml of N,N-dimethylformamide (peptide synthesis grade , Biosolve, Valkenswaard, NL), 204 μl of N,N'-diisopropylcarbodiimide was added. After incubation for 30 min at 21 °C, 1 g Amino-HES10/0.4 was added. After shaking at 22°C for 19 hours, the reaction mixture was added to 84 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, redissolved in 50 ml of water, dialyzed against water for 2 days (SnakeSkin dialysis tube, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and lyophilized. the

Example 1.4: Synthesis of aldehyde-functionalized hydroxyethyl starch from hydroxyethyl starch and formylbenzoic acid

Oxo-HES10/0.7 (MW=10kD, DS=0.7) was prepared by Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. the

Take 83 mg of 4-formylbenzoic acid and 180 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) dissolved in 5 mL of N,N-dimethylformamide (DMF, peptide Synthetic grade, Biosolve, Valkenswaard, NL), 78 μl of N,N'-diisopropylcarbodiimide was added. After incubation at 21 °C for 30 min, 0.5 g of oxo-HES10/0.7 was added. After shaking at 22°C for 19 hours, the reaction mixture was added to 37.5 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, redissolved in a mixture of 2.5 ml water and 2.5 ml DMF, and precipitated once more as described above. The reaction product was collected by centrifugation as described above, redissolved in 10 ml of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and lyophilized. the

Example 1.5: Synthesis of aldehyde-functionalized hydroxyethyl starch from hydroxyethyl starch and formylbenzoic acid

HES10/0.7 (MW=10 kD, DS=0.7) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D. the

Take 50 mg of 4-formylbenzoic acid and 108 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) dissolved in 3 mL of N,N-dimethylformamide (peptide synthesis grade , Biosolve, Valkenswaard, NL), add 47 μl N, N'-diisopropylcarbodiimide. After incubation at 21°C for 30 min, 0.3 g of HES10/0.7 was added. After shaking at 22°C for 19 hours, the reaction mixture was added to 23 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, redissolved in a mixture of 1.5 ml water and 1.5 ml DMF, and precipitated once more as described above. The reaction product was collected by centrifugation as described above, redissolved in 10 ml of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and lyophilized. the

Example 1.6: Synthesis of aldehyde-functionalized hydroxyethyl starch from amino-functionalized hydroxyethyl starch and pentafluorophenyl formylbenzoate

Oxo-HES10/0.7 (MW=10 kD, DS=0.7) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE19628705A1. the

Take 6.0 g (0.6 mmol) of oxo-HES10/0.7 dissolved in 20 ml of anhydrous dimethyl sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D), and add dropwise to 6 ml (60 mmol) under nitrogen , 4-diaminobutane in 11 ml of anhydrous dimethyl sulfoxide solution, stirred at 40 ° C for 19 hours. The reaction mixture was added to a mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was centrifuged, washed with a mixture of 20 ml ethanol and 20 ml acetone, redissolved in 80 ml water, and the solution was dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) , and then freeze-dried. Obtained 52% (3.15 g) Amino-HES10/0.7. the

Pentafluorophenyl 4-formylbenzoate was synthesized as described in J.S. Lindsey et al., Tetrahedron 50 (1994) pp.8941-68, especially p.8956. Take 50mg amino-HES10/0.7 and dissolve in 0.5ml N,N-dimethylformamide (peptide synthesis grade, Biosolve, Valkenswaard, NL), and add 15.3mg pentafluorophenyl 4-formylbenzoate. After shaking for 22 hours at 22°C, the reaction mixture was added to 3.5 ml of ice-cold 2-propanol. The precipitated product was collected by centrifugation at 4 °C, washed with 4 ml of ice-cold 2-propanol, redissolved in 50 ml of water, and dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D ) and freeze-dried. the

Example 1.7: Synthesis of aldehyde-functionalized hydroxyethyl starch from hydroxyethyl starch and pentafluorophenyl formylbenzoate

Oxo-HES 10/0.7 (MW=10kD, DS=0.7) was prepared by Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. Pentafluorophenyl 4-formylbenzoate as J.S.Lindsey et al., Tetrahedron50 (1994) pp.8941-68, especially the synthesis described on p.8956. Take 200mg of oxo-HES10/0.7 and dissolve in 2ml of N,N-dimethylformamide (peptide synthesis grade, Biosolve, Valkenswaard, NL), and add 61.2mg of pentafluorophenyl 4-formylbenzoate. After shaking at 22°C for 22 hours, the reaction mixture was added to 15 ml of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, redissolved in a mixture of 1.4 ml water and 0.7 ml DMF, and precipitated once more as described above. The reaction product was collected by centrifugation as described above, redissolved in 10 ml of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and lyophilized. the

Example 1.8: Synthesis of aldehyde-functionalized hydroxyethyl starch from amino-functionalized hydroxyethyl starch and 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid

Oxo-HES10/0.4 (MW=10kD, DS=0.4) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. the

Take 5.1 g (0.51 mmol) of oxo-HES10/0.4 dissolved in 15 ml of anhydrous dimethyl sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D), and add dropwise to 5.1 ml (51 mmol) under nitrogen A solution of 1,4-diaminobutane in 10 ml of anhydrous dimethyl sulfoxide was stirred at 40°C for 19 hours. The reaction mixture was added to a mixture of 80 ml ethanol and 80 ml acetone. The resulting precipitate was separated by centrifugation, washed with a mixture of 20 ml of ethanol and 20 ml of acetone, and redissolved in 80 ml of water. The solution was dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) and then lyophilized. 67% (3.4 g) of amino-HES 10/0.4 were obtained. the

Take 80.5mg 4-(4-formyl-3,5-dimethoxyphenoxy)butanoic acid (Calbiochem-Novabiochem, Laufelfingen, CH) and 61mg 1-hydroxy-1H-benzotriazole (Aldrich, Sigma -Aldrich Chemie GmbH, Taufkirchen, D) dissolved in 3ml N,N-dimethylformamide (peptide synthesis grade, Biosolve, Valkenswaard, NL), added 45.4 μl N,N'-diisopropylcarbodiimide . After incubation at 21 °C for 30 min, 0.3 g Amino-HES 10/0.4 was added. After shaking at 22°C for 22 hours, the reaction mixture was added to 23 ml of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, redissolved in a mixture of 2 ml water and 1 ml DMF, and precipitated once more as described above. The reaction product was collected by centrifugation as described above, redissolved in 10 ml of water, dialyzed against water for 1 day (SnakeSkin dialysis tubing, cut-off value 3.5 kD, PerbioSciences Deutschland GmbH, Bonn, D), and lyophilized. the

Embodiment 2 : Synthesis of G-CSF conjugates by reductive amination

Example 2.1(a): Synthesis of a G-CSF-conjugate by reductive amination using hydroxyethyl starch with non-oxidized reducing ends at pH=7.4 (comparative example)

In Example 2.1, an attempt was made to prepare a HES-G-CSF conjugate using the synthesis method of WO 03/074087 (Example 12, p.22-23). the

来自Amgen，Munchen，D或 

来自Aventis Pharma AG，Zurich，CH，分别为3mg/ml)的0.1M磷酸钠缓冲液pH 7.4水溶液中添加3.33μl HES10/0.4(MW＝10kD，DS＝0.4，Suprarnol Parenteral Colloids GmbH，Rosbach-Rodheim，D，79mg/ml)的相同缓冲液的溶液。向该混合物中添加3.33μl 60mM氰基硼氢化钠的相同缓冲液的溶液，所得混合物在22℃下孵育4小时。然后，再添加3.33μl新鲜制备的60mM氰基硼氢化钠溶液。在30小时孵育期间，共添加5份3.33μl新鲜制备的60mM氰基硼氢化钠溶液。通过凝胶电泳分析反应混合物。未观察到反应。  To 3.33μl G-CSF (

from Amgen, Munchen, D or

3.33 μl of HES10/0.4 (MW=10 kD, DS=0.4, Suprarnol Parenteral Colloids GmbH, Rosbach-Rodheim, D, 79 mg/ml) solution in the same buffer. To this mixture was added 3.33 μl of a solution of 60 mM sodium cyanoborohydride in the same buffer, and the resulting mixture was incubated at 22° C. for 4 hours. Then, an additional 3.33 μl of freshly prepared 60 mM sodium cyanoborohydride solution was added. During the 30 h incubation, a total of 5 3.33 μl aliquots of freshly prepared 60 mM sodium cyanoborohydride solution were added. The reaction mixture was analyzed by gel electrophoresis. No response was observed.

Example 2.1(b): Synthesis of G-CSF-conjugates by reductive amination using hydroxyethyl starch with non-oxidized reducing ends at pH = 5.0 to 9.2 (comparative example)

To 3.33 μL of G-CSF (G-CSF from Strathmann Biotec AG, Hamburg, D, 3 mg/mL) in the indicated buffer in water was added 3.33 μL of HES (300 mg/ml) in the same buffer solution. The mixture was cooled to 4°C, 3.33 μL of 60 mM sodium cyanoborohydride in the same buffer solution was added at 4°C, and the resulting mixture was incubated at 4°C for 20 hours. the

Use the following HES preparations and buffers:

a) Buffer: 0.1M sodium acetate buffer pH 5.0

-HES10/0.4 (MW=10kD, DS=0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

-HES50/0.4 (MW=50kD, DS=0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

-HES50/0.7 (MW=50kD, DS=0.7, Supramol ParenteralColloids GmbH, Rosbach-Pvodheim, D)

b) Buffer: 0.1M sodium phosphate buffer pH 7.2

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

c) Buffer: 0.1M sodium borate buffer pH 8.3

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

d) Buffer: 0.2M potassium borate buffer pH 9.2

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

Each reaction mixture was analyzed by gel electrophoresis. Coupling reactions were not observed or were almost negligible (gel scans of reactions b) to d) not shown). the

Example 2.2 : Synthesis of GC SF-conjugates at pH 5.0 to 9.2 by reductive amination using hydroxyethyl starch with oxidized reducing ends (comparative example)

To 3.33 μL of G-CSF (G-CSF from Strathmann Biotec AG, Hamburg, D, 3 mg/mL) in the indicated buffer in water was added 3.33 μL of oxo-HES (300 mg/ml) in the same buffer solution. The mixture was cooled to 4°C, 3.33 μL of 60 mM sodium cyanoborohydride solution in the same buffer was added at 4°C, and the resulting mixture was incubated at 4°C for 17 hours. the

Use the following HES preparations and buffers:

a) Buffer: 0.1M sodium acetate buffer pH 5.0

-Oxo-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

-Oxo-HES50/0.4 (MW=50kD, DS=0.4, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D)

-Oxo-HES50/0.7 (MW=50kD, DS=0.7, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D)

b) Buffer: 0.1M sodium phosphate buffer pH 7.2

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

c) Buffer: 0.1M sodium borate buffer pH 8.3

-HES10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

d) Buffer: 0.2M potassium borate buffer pH 9.2

-HES 10/0.7 (MW=10kD, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D)

Each reaction mixture was analyzed by gel electrophoresis. Coupling reactions were not observed or were almost negligible (gel scans of reactions b) to d) not shown). the

Oxidation of HES10/0.4 (MW=10 kD, DS=0.4) was performed by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. the

Example 2.3: Synthesis of G-CSF-conjugates by reductive amination using aldehyde-functionalized hydroxyethyl starch synthesized via periodate oxidation

来自Aventis Pharma AG，Zurich，CH和 

来自Amgen，Munchen，D，分别3mg/mL)的0.1M乙酸钠缓冲液pH 5.0水溶液中添加3.33μl醛基-HES(79mg/mL)的相同缓冲液溶液。向混合物中添加3.33μL 60mM氰基硼氢化钠的相同缓冲液溶液，混合物在21℃下孵育25小时。通 过凝胶电泳分析反应混合物。  To 3.33μl G-CSF (

from Aventis Pharma AG, Zurich, CH and

To 0.1 M sodium acetate buffer pH 5.0 aqueous solution from Amgen, Munchen, D, respectively 3 mg/mL) was added 3.33 μl of aldehyde-HES (79 mg/mL) in the same buffer solution. To the mixture was added 3.33 μL of a 60 mM sodium cyanoborohydride solution in the same buffer, and the mixture was incubated at 21° C. for 25 hours. The reaction mixture was analyzed by gel electrophoresis.

Use the following aldehyde functionalized HES conjugates:

根据上述实施例1.1(a)制备；  (iN) use

According to above-mentioned embodiment 1.1 (a) preparation;

(ii-N) use According to the above-mentioned embodiment 1.1 (b) preparation;

根据上述实施例1.2(a)制备；  (iii-N) use

According to the above-mentioned embodiment 1.2 (a) preparation;

根据上述实施例1.2(b)制备；  (iv-N) use

According to the above-mentioned embodiment 1.2 (b) preparation;

根据上述实施例1.1(a)制备；  (iG) use

According to above-mentioned embodiment 1.1 (a) preparation;

根据上述实施例1.1(b)制备；  (ii-G) use

According to the above-mentioned embodiment 1.1 (b) preparation;

根据上述实施例1.2(a)制备；  (iii-G) use

According to the above-mentioned embodiment 1.2 (a) preparation;

根据上述实施例1.2(b)制备。  (iv-G) use

Prepared according to Example 1.2(b) above.

Example 2.4: Synthesis of G-CSF-conjugates by reductive amination using aldehyde-functionalized hydroxyethyl starch synthesized by coupling hydroxyethyl starch with formyl carboxylic acid

To 3.33 μl of G-CSF (G-CSF from Strathmann Biotec AG, Hamburg, D, 3 mg/ml) in 0.1 M sodium acetate buffer pH 5.0 in water was added 3.33 μl of aldehyde-HES (118.5 mg/mL) in the same buffer Liquid solution, cooled to 4 °C. At 4°C, 3.33 μl of 60 mM sodium cyanoborohydride in the same buffer solution was added to the mixture, and the mixture was incubated at 4°C for 17 hours. The reaction mixture was analyzed by gel electrophoresis. the

Use the following aldehyde functionalized HES conjugates:

(v) prepared according to the above-mentioned embodiment 1.4;

(vi) prepared according to the above-mentioned embodiment 1.5;

(vii) prepared according to the above-mentioned embodiment 1.6;

(viii) prepared according to the above-mentioned embodiment 1.7;

(ix) Prepared according to Example 1.8 above. the

Example 2.5: Synthesis of G-CSF-conjugates by reductive amination using aldehyde-functionalized hydroxyethyl starch coupled with formyl carboxylic acid

To 2.5 ml of G-CSF (G-CSF from Strathmann Biotec AG, Hamburg, D, 2.27 mg/ml) in 0.1 M sodium acetate buffer pH 5.0 in water was added 136 mg of aldehyde-HES prepared as described above in Example 1.3 10/0.4, the solution was cooled to 0°C. To the mixture was added 2.5 ml of an ice-cold 40 mM sodium cyanoborohydride solution in the same buffer, and the mixture was incubated at 4°C for 17 hours. The reaction mixture was analyzed by gel electrophoresis. the

Embodiment 3 : Synthesis of G-CSF conjugates by SH alkylation

Oxo-HES10/0.7 (MW=10kD, DS=0.7) was prepared by Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. the

Dissolve 6.0 g (0.6 mmol) of oxo-HES10/0.7 in 20 ml of anhydrous dimethyl sulfoxide (DMSO, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D), and add dropwise to 6 ml (60 mmol) under nitrogen A solution of 1,4-diaminobutane in 11 ml of anhydrous dimethyl sulfoxide was stirred at 40°C for 19 hours. The reaction mixture was added to a mixture containing 80 ml ethanol and 80 ml acetone. The resulting precipitate was separated by centrifugation, washed with a mixture containing 20 ml of ethanol and 20 ml of acetone, and redissolved in 80 ml of water. The solution was dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D) and then lyophilized. Obtained 52% (3.15 g) Amino-HES10/0.7. the

来自Aventis Pharma AG，Zurich，CH，分别3μg/μl，溶于磷酸盐缓冲液中)，混合物在25℃下孵育16小时。真空浓缩后，通过凝胶电泳分析反应混合物。  Dissolve 132 μg Amino-HES 10/0.7 in 100 μl sodium phosphate buffer (0.1M, 0.15M NaCl, 50 mM EDTA, pH 7.2), add 10 μl 17.5 mg/ml N-α (maleimidoacetoxy) Succinimidyl esters (AMAS) in anhydrous DMSO (both from Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were incubated at 25°C for 80 minutes and then at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D), centrifuge and filter at 13,000rpm to remove excess AMAS, wash twice with 450μl phosphate buffer for 30 minutes, and wash once with 450μl buffer B. Add 10 μg G-CSF ( from Amgen, Munchen, D and

from Aventis Pharma AG, Zurich, CH, 3 μg/μl, respectively, in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After concentration in vacuo, the reaction mixture was analyzed by gel electrophoresis.

Choose from the following methods:

)采用磷酸钠缓冲液(0.1M，0.15MNaCl，50mM EDTA，pH 7.2)作为缓冲液B。  (x)G-CSF(

) using sodium phosphate buffer (0.1 M, 0.15 M NaCl, 50 mM EDTA, pH 7.2) as buffer B.

)采用磷酸钠缓冲液(0.1M，0.15MNaCl，50mM EDTA，pH 7.2)作为缓冲液B。  (xi)G-CSF(

) using sodium phosphate buffer (0.1 M, 0.15 M NaCl, 50 mM EDTA, pH 7.2) as buffer B.

)采用磷酸钠缓冲液(0.1M，0.15MNaCl，50mM EDTA，pH 7.2)与8M尿素，1％SDS，pH 7.4的1∶1(v/v)混合物作为缓冲液B。  (xii) G-CSF (

) using a 1:1 (v/v) mixture of sodium phosphate buffer (0.1M, 0.15M NaCl, 50mM EDTA, pH 7.2) and 8M urea, 1% SDS, pH 7.4 as buffer B.

)采用磷酸钠缓冲液(0.1M，0.15MNaCl，50mM EDTA，pH 7.2)与8M尿素，1％SDS，pH 7.4的1∶1(v/v)混合物作为缓冲液B。  (xiii) G-CSF (

) using a 1:1 (v/v) mixture of sodium phosphate buffer (0.1M, 0.15M NaCl, 50mM EDTA, pH 7.2) and 8M urea, 1% SDS, pH 7.4 as buffer B.

)采用8M尿素，1％SDS，pH 7.4作为缓冲液B。  (xiv)G-CSF(

) using 8M urea, 1% SDS, pH 7.4 as buffer B.

)采用8M尿素，1％SDS，pH 7.4作为缓冲液B。  (xv)G-CSF(

) using 8M urea, 1% SDS, pH 7.4 as buffer B.

Embodiment 4 : Synthesis of G-CSF conjugates by reacting hydroxyethyl starch with reactive ester groups and G-CSF

Example 4.1:

Oxo-HES10/0.4 (MW=10, 559D, DS-0.4) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. The degree of oxidation of oxo-HES is 95%. the

Take 66 mg of oxo-HES10/0.4 and dissolve in 0.5 ml of anhydrous DMF. To this solution was added 3.4 mg of N,N'-disuccinimidyl carbonate, and the mixture was stirred at room temperature for 2 hours. The resulting solution had a reactive HES concentration of 13% by weight. the

Take a G-CSF solution (Strathmann BiotecAG, Hamburg, D) with a concentration of about 0.5mg G-CSF/ml, use a cooling centrifuge, perform ultracentrifugation at a cutoff value of 10kD, and concentrate to a concentration of 10mg/ml. the

To 0.5 ml of this concentrated G-CSF solution was added 180 μl of sodium bicarbonate solution. Then, the reactive HES solution was added dropwise to the protein solution in 3 portions (100 μl each time) until the reaction ended after about 30 minutes. Therefore, the total molar ratio of reactive HES:G-CSF is 20:1. Then, the pH of the mixture was adjusted to 4.0 using 0.1N HCl. the

HPGPC analysis (High Performance Gel Permeation Chromatography) gave about 70% yield. The results are shown in FIG. 9 . the

The mixture can be stored at pH 4.0 and 4°C for 4 days and remains stable, i.e. unchanged, according to HPGPC analysis. the

A 10Kd ultrafiltration membrane can be used to reduce the content of reaction by-products in the mixture, such as unreacted oxo-HES and free N-hydroxysuccinimide and solvent, without difficulty in a cooled centrifuge. The results of this reduced content experiment are shown in FIG. 10 . the

Example 4.2:

Oxo-HES10/0.4 (MW=10, 559D, DS=0.4) was prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; according to DE 19628705A1. The degree of oxidation of oxo-HES is 95%. the

Take 400mg of oxo-HES10/0.4 and dissolve in 1ml of anhydrous DMF. To this solution was added 21 mg of N,N'-disuccinimidyl carbonate, and the mixture was stirred at room temperature for 2 hours. The resulting solution had a reactive HES concentration of 40% by weight. the

Take a solution of G-CSF (Strathmann BiotecAG, Hamburg, D) with a concentration of about 0.5mg G-CSF/ml, use a cooling centrifuge, perform ultracentrifugation at a cutoff value of 10kD, and concentrate to a concentration of 10mg/ml. the

To 0.5 ml of this concentrated G-CSF solution was added 180 μl of sodium bicarbonate solution. Then, the reactive HES solution was added dropwise to the protein solution in 3 portions (100 μl each time) until the reaction ended after about 30 minutes. Therefore, the total molar ratio of reactive HES:G-CSF is about 50:1. Then, the pH of the mixture was adjusted to 4.0 using 0.1N HCl. the

HPGPC analysis (High Performance Gel Permeation Chromatography) gave greater than 95% yield. Unreacted G-CSF was not detected. The results are shown in FIG. 11 . the

The mixture can be purified without difficulty using ultrafiltration techniques. The results are shown in FIG. 12 . the

Example 5

5.1. Purification

(Amgen)相同特性的纯化G-CSF，取其中一份保留不修饰，作为对照。  Get basically has with commodity

(Amgen) purified G-CSF with the same characteristics, one of which was kept unmodified as a control.

5.2 Synthesis of conjugates of HES and G-CSF

Essentially as described in Example 4.2, but where oxo-HES50/0.7 (Sample No. A60) was used instead, or the conjugate was synthesized as described in Example 2.5 (Sample No. A32) and used for further buffer exchange and purification. the

5.3 Buffer exchange between G-CSF and HES-modified G-CSF samples using anion exchange chromatography before purification

HES-modified G-CSF samples or unmodified G-CSF (as a control) (0.5-5 mg protein) were taken for buffer exchange using a Vivaspin 6 concentrator unit (10.000 MWCO PES, Vivascience, cat. no. VS0602). Samples were concentrated to 0.5-0.7ml and diluted to 5ml with 10mM sodium phosphate buffer pH 7.2. Each sample was subjected to this concentration/buffer exchange cycle a total of 3 times. the

Anion of 5.4G-CSF and its HES-modified form on DEAE-Sepharose column

exchange chromatography

Purified HES-modified G-CSF samples and unmodified G-CSF samples for control were analyzed by ion-exchange chromatography using an AKTA explorer 10 system at room temperature. An aliquot of G-CSF before or after HESylation was dialyzed against the buffer A (10 mM sodium phosphate, pH 7.2) by ultrafiltration, or diluted with approximately 13 volumes of buffer A. The column contained 2 ml of DEAE-Sepharose (DEAE-Sepharose CL-6B, Pharmacia Cat. No. 17-0710-01) with the addition of 5.0 column volumes (CV) of 6.5M Guanidine/HCl, 5.0CV Buffer A, 5.0CV Buffer C (1.5M NaCl in 10mM sodium phosphate solution, pH 7.2), followed by 10CV buffer A for regeneration. Samples (0.8-4.5 ml in 10 mM sodium phosphate buffer pH 7.2) were then injected at a flow rate of 0.6 ml/min. The sample loop was then washed with 10ml (when using a 2ml sample loop) or 20ml (when using a 5ml sample loop) of buffer A, and the column was washed again with 0-22CV of buffer A depending on the sample added (flow rate = 0.8ml /min). Elution was performed at a flow rate of 0.6 ml/min using a linear gradient of 5 CV from 0-100% buffer B and 2.5 CV isocratic 100% buffer B. The column was re-equilibrated with 5 CV of buffer A and regenerated using a flow rate of 1 ml/min as described above. the

When required, samples were concentrated using Vivaspin concentrators and buffer exchanged as described above. Samples were stored at 0-8°C in 10 mM sodium acetate buffer pH 4.0 before or after sterile filtration using a 0.2 μm filter unit (Corning, cat. no. 431215). The following samples were prepared for in vitro bioassay and further analysis. Protein concentrations were determined as described in section 6.1 below:

I.0401-15/A33, 0.44mg/ml, volume = 500μl

G-CSF (Escherichia coli (E.coli))

II.0402-03/A60, 0.35mg/ml, volume = 600μl

H-G-CSF (G-CSF HES modification, 10/0.4)

III.0401-13/A32, 0.28mg/ml, volume = 900μl

G-CSF (Escherichia coli) HES modification, 10/0.4

IV.0401-28/A58, 0.60mg/ml, volume = 350μl

Neupogen

V.0401-28/A57, 0.50mg/ml, volume = 400μl

Neulasta

5.5. Further analysis of G-CSF samples

An aliquot of the sample was taken to analyze its protein content and modification. the

5.5(a) G-CSF protein quantification by RP-HPLC

The G-CSF protein content of the samples was quantified using an unmodified protein preparation (concentration: 0.453 mg/ml) as a standard. the

Adopt Dionex HPLC system, which includes pump P 680A HPG, degassing unit Degasys DG 1210, autosampler and injector ASI-100, sample loop 250 μL, column thermostat TCC 100 and UV/Vis-detector UVD170U, which is equipped with Software Chromeleon chromatography management system. A pre-packed column CC 8/4 Nucleosil 120-5C4, Macherey-Nagel, Cat. No. 721889 and a separation column 40 C-4 Nucleosil MPN, 5 μm, 125×4 mm RP-column (Macherey-Nagel, order No. 720045.40) were used. Solvent A is H ₂ O plus 0.06% (v/v) trifluoroacetic acid, solvent B is 90% acetonitrile aqueous solution containing 0.06% (v/v) trifluoroacetic acid, flow rate: 1ml/min. UV detection was performed at wavelengths of 214, 221, 260 and 280 nm.

About 10-20 μg of sample was injected into the RP-HPLC column. The following gradients are used:

0-5 minutes: 0-10% B

-17 minutes: 10-45% B

-35 minutes: 45-80% B

-36 minutes: 80-100% B

-38 minutes: 100% B

-39 minutes: 10% B

-45 minutes: 10% B

The peak area obtained using the elution position of the standard G-CSF preparation was compared to a reference standard obtained at a wavelength of 280 nm at approximately 29 minutes. the

5.5(b) Reduction of G-CSF protein + carboxamidomethylation

Aliquots of G-CSF protein samples were reduced and carboxamidomethylated as described (Guillermina Forno, Mariela Bollati Fogolin, Marcos Oggero, Ricardo Kratje, Marina Etcheverrigaray, Harald S. Conradt, Manfred Nimtz (2004) N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cellline; European J. Biochem, 273(5), 907-919). Carboxamidomethylation produces modified cysteine residues. Carboxamidomethylated proteins were subjected to endoprotease Glu-C digestion at an enzyme/substrate ratio of 0.2: ₁₀ in 25 mM _NH4HCO3 containing 1 M urea, pH 7.8, for 18-24 hours.

5.5(c) Separation of endo-Glu-C peptides by RP-HPLC

Peptides resulting from endo-Endo-Glu-C digestion were separated on a Dionex HPLC system consisting of pump P 680A HPG, degassing unit Degasys DG 1210, autosampler and syringe ASI-100, sample loop 250 μL, column thermostat TCC 100 and UV/Vis-detector UVD 170U with software Chromeleon chromatography management system installed in it. A pre-packed column CC 8/4 Nucleosil 120-5C4, Macherey-Nagel, Cat. No. 721889 and a separation column 40C-4 Nucleosil MPN, 5 μm, 125×4 mm RP-column (Macherey-Nagel, order No. 720045.40) were used. Solvent A is H ₂ O plus 0.06% (v/v) trifluoroacetic acid, solvent B is 90% acetonitrile aqueous solution containing 0.06% (v/v) trifluoroacetic acid, flow rate: 1ml/min. The following gradients are used:

0-5 minutes: 10% B

-17 minutes: 45% B

-65 minutes: 100% B

-67 minutes: 100% B

-69 minutes: 10% B

-75 minutes: 10% B

UV detection was performed at wavelengths of 214, 221, 260 and 280 nm. Peptides resulting from endo-Glu-C digestion were isolated (data not shown). the

5.5(d) Analysis of Proteolytic Peptides by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The intact N-terminus of G-CSF in the different samples prepared was detected by mass spectrometry. Samples (3-5 μg) of the reduced and carboxamidomethylated protein samples digested with the endoprotease Glu-C were used directly for MS-analysis (excluding RP-HPLC in step 6.3) with C18-containing reverse substance ZipTip pipette tips were purified according to the manufacturer's instructions. After washing with 0.1% (v/v) formic acid, elution of the peptide was performed with 10 μl of 0.1% (v/v) formic acid in 60% (v/v) acetonitrile. the

The peptide fragments of proteolysis (endo-Glu-C) were analyzed with a Bruker ULTRAFLEX time-of-flight (TOF/TOF) instrument in linear positive ion mode, using 22.4 mg of 3,5-dimethoxy-4-hydroxy-cinnamic acid in A solution in 400 μL acetonitrile and 600 μl 0.1% (v/v) trifluoroacetic acid in water was used as a substrate; and a solution containing 19 mg α-cyano-4-hydroxycinnamic acid in the same solvent mixture was used as a substrate, and a reflector (reflectron ) enhanced resolution, determination of (glycosyl)-peptides. Take 1 μL of the sample solution with a concentration of about 1-10 pmol-μl-1 and mix it with an equal amount of each matrix. The mixture was spotted on a stainless steel target and analyzed after drying at room temperature. Mass spectra were recorded in the mass range 900-5000 Daltons. The table below shows the correlation of expected mass with each G-CSF peptide. the

Table: Theoretical (monoisotopic) masses of endo-Glu-C peptides produced by XM02

Mass (Daltons) Observations from this study aa position artificial modification peptide sequence 2132.11 + 1-20 Cys_CAM: pos 18 m/z 2189.13 MTPLGPASSLPQSFLLKCLE 1512.81 + 21-34 ----- QVRKIQGDGAALQE 1534.74 + 35-47 Cys_CAM: pos 37, 43 m/z 1648.78 KLCATYKLCHPEE 4942.63 - 48-94 Cys_CAM: pos 65, 75 m/z 5056.68 LVLLGHSLGIPWAPLSSSCPS QALQLAGCLSQLHSGLFLYQ GLLQALE 502.25 - 95-99 ----- GISPE 2835.37 - 100-124 ----- LGPTLDTLQLDVADFATTIW QQMEE 4026.08 + 125-163 ----- LGMAPALQPTQGAMPAFASA FQRRAGGVLVASHLQSFLE 1438.83 + 164-175 ----- VSYRVLRHLAQP

Cysteine residues were carboxamidomethylated; fat-labeled peptides were detected on the MALDI/TOF spectrum of unmodified G-CSF. the

The N-terminal endo-Glu-C peptide (MTPLGPASSLPQSFLLKCLE; m/z 2189.1), including protein sites 1-20, was detected in the MALDI /TOF-MS spectrum detection. the

5.6. Results

5.6(a) Purification of G-CSF and HES modified variants

A32, A60 and unmodified G-CSF were purified using DEAE-Sepharose CL-6B column as described in A4. the

For the unmodified sample 0401-15/A33, no significant absorption was detected at 280nm in the flow-through, and the specificity of the protein eluted at 280nm with 40-50% buffer B (0.16-0.20M NaCl) in a volume of 6ml The peak area is 660mAU×ml×mg ^-1 .

Sample 0401-14/A32 (derived from 0401-15/A33; HESylated using aldehyde HES10/0.4) was run over a large gradient range of concentrations 20-80% (0.08-0.32M NaCl) in buffer B and a volume of 12ml elute. At 280nm, about 90% of the total peak area was detected in the flow-through, which contained about 50% of the total protein, detected by SDS-PAGE analysis as shown in Figure 13 above, and its molecular weight was apparently slightly higher than that of the washed out of protein. the

Sample 0402-03/A60 (HES10/0.4HESylated, following the full procedure of Example 4.2) eluted at a similar concentration of 20-80% buffer B in a volume of 10.5 ml. At this time, about 35% of the total peak area was detected in the flow-through at 280 nm, however, no unbound protein was detected in this fraction by SDS-PAGE analysis. Compared to the specific peak area of sample 0401-15/A33, the protein content in the eluate of sample 0402-03/A60 was 45% higher than the specified amount of protein applied to the column. the

Protein recovery was calculated based on the peak area (A280nm) of the eluted fractions compared to unmodified G-CSF protein. the

Table 1: Comparison of peak areas detected at 280nm

DEAE-Sepharose operation number illustrate Calculated protein injection volume Eluate area [280nm] (mAU×ml) Yield of eluate compared to eluate of DS01 A33** DS01A33 G-CSF 0.5mg 330 (0.50mg) DS03A32 Hesified HES 10/0.4 4.0 mg 1560 2.36mg DS04A60 Hesified HES 10/0.4 0.9mg 870 1.32mg

The RP-HPLC quantification of ** protein confirms these results

5.6(b) Peptide mapping and protein analysis by MALDI/TOF MS after endoprotease Glu-C treatment

The N-terminus of both carboxamidomethylated unmodified G-CSF (Figure 14) and the commercially available product Neupogen (data not shown) was clearly detected by MALDI/TOF-MS after digestion with the endoprotease Glu-C Peptide (MTPLGPASSLPQSFLLKC*LE, m/z 2189.1; cysteines are carboxamidomethylated). This signal was absent in samples HES-modified by reductive amination (Figure 15) and Neulasta (data not shown), indicating that the peptide was modified. For HES-modified G-CSF, wherein the modification was performed by activated ester chemistry, the relative signal intensity of the N-terminal peptide was detected relative to the unmodified starting material A32 (Figure 16), indicating that the HES modification of this derivative has Realized on different amino acid side chains. the

N-terminal sequencing of HES-modified G-CSF (sample A33 and commercial product Neulasta) showed that its N-terminal was blocked, suggesting that the N-terminal methionine residue of the protein derivative was modified by the HES derivative. Since no signal corresponding to a peptide comprising amino acid residue positions 35-47 (KLCATYKLCHPEE; both cysteine residues carboxamidomethylated, m/z 1648.78) was detected in sample A60, the It was concluded that one or two of the lysine residues (positions 35 and 41) might be modified by HES. the

references:

Guillermina Forno，Mariela  Bollati Fogolin，Marcos Oggero，Ricardo Kratje，Marina Etcheverrigaray，Harald S.Conradt，ManfredNimtz(2004)N-and O-linked carbohydrates and glycosylation siteoccupancy in recombinant human granulocyte-macrophagecolony-stimulating factor secreted by a Chinese hamster ovary cellline; European J. Biochem, 271(5), 907-919

Nimtz, M., Grabenhorst, E., Conradt, H.S., Sanz, L. & Calvete, J.J. (1999) Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP-I andPSP-II glycoforms. Eur. Biochem. 265, 703-718.

Nimtz, M., Martin, W., Wray, V., Kloppel, K.-D., Agustin, J. & Conradt, H.S. (1993) Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cell. Eur J. Biochem, 213, 39-56

S.，Derr，P.，Conradt，U.S.，Nimtz，M.，Hale，G.& Kirchhoff，C.(1999)Male-specific modification of human CD52.J.Biol.Chem.274，29862-29873  Nimtz, M., Noll G. Paques, E. & Conradt, HS (1990) Carbohydrate Structures of human tissue plasminogen activator variant expressed inrecombinant Chinese hamster ovary cells. FEBS Lett.271, 14-18.

S., Derr, P., Conradt, US, Nimtz, M., Hale, G. & Kirchhoff, C. (1999) Male-specific modification of human CD52. J. Biol. Chem. 274, 29862-29873

E.Grabenhorst and H.S.Conradt(1999) The Cytoplasmic, Transmembrane and the Stem Regions of G1ycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi.J.Biol.Chem., 274, 36107-36116

E.Grabenhorst, A.Hoffmann, M.Nimtz, G.Zett1meiβ1 and HSConradt (1995) Construction of stable BHK-21 cell coexpressing human secretory glycoproteins and human Galβ1-4GlcNAc-R 2,6-sialyltransferase: 2,6-linked NeuAc is preferably attached to the Galβ1-4GlcNAcβ1-2ManB1-3-branch of biantennaryoligosaccharides from secreted recombinant-trace protein. Eur.J.Biochem., 232 718-725

Example 6 : In vitro results of G-CSF-conjugates prepared in Examples 2.5 and 4.2 and purified according to Example 5: Mitogenicity of G-CSF variants on mouse NFS-60 cells

G-CSF is known to have specific effects on the proliferation, differentiation and activation of hematopoietic cells of the neutrophil lineage. The mitogenic ability of G-CSF variants was tested using mouse NFS-60 cells (N. Shirafuji et al., Exp. Hematol. 1989, 17, 116-119). Cells were grown in RPMI medium (Gibco INVITROGEN GmbH, Gibco INVITROGEN GmbH, Karlsruhe), collected by centrifugation, washed, and divided into 100,000 cells per well in a 24-well plate. Cells were adapted for 1 hour at 37°C in RPMI medium without WEHI-3B conditioned medium, and then G-CSF growth factor samples diluted in the same medium were added. NFS-60 cells were exposed to purified G-CSF variants for 3 days at 37°C, after which cells were electronically counted (Casy TT cell counter, Scharfe System, Reutlingen, D). The results are shown in Figure 12. As can be seen from Figure 12, the different G-CSF variants (0.5-50 pg/ml) could stimulate an increase in cell number after 3 days compared to the medium without growth factor addition. the

The unmodified control protein G-CSF/A33 and G-CSF/A58 stimulated the cells to a very similar degree (ED ₅₀ =5-10pg/ml), while the G-CSF conjugate G-CSF/A60G-CSF/A32 And G-CSF/A57 showed only slightly lower activity than the unmodified type (ED ₅₀ =10-25 pg/ml) (see Fig. 8 ).

Example 7 Synthesis of G-CSF-conjugates

Example 7.1. Synthesis of Aldehyde-HES Derivatives

Example 7.1.(a) Synthesis of Amino HES10/0.4

Take 5.12 g of oxo-HES10/0.4 (MW=10000D, DS=0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, according to DE 19628705A1) and heat it under vacuum at 80°C overnight, dissolve it in 25 mL of anhydrous dimethylformamide under nitrogen Sulfoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added with 5.13 mL of 1,4-diaminobutane. After stirring at 40° C. for 17 hours, the reaction mixture was added to 150 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, washed with 40 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol, and collected by centrifugation. The crude product was dissolved in 80 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of isolated product was 67%. the

Example 7.1 (b) Synthesis of Aldehyde HES10/0.4

105 mg of 4-formylbenzoic acid and 135 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 7 mL of N,N-dimethylformamide (peptide Synthetic grade, Biosolve, Valkenswaard, NL), 135 μL of N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkrchen, D) was added. After incubation at 21°C for 30 min, 0.7 g Amino HES10/0.4 (synthesized as described in 1.1) was added. After shaking at 22°C for 18 hours, the reaction mixture was added to 42 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4 °C, redissolved in 5 mL of DMF, and precipitated using 42 mL of ethanol/acetone as described above. After centrifugation, the collected precipitate was dissolved in water, dialyzed against water for 1 day SnakeSkin dialysis tubes, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of the isolated product was 95%. the

Embodiment 7.1 (c) synthesis of amino HES10/0.7

6.02 g of oxo-HES10/0.7 (MW=10000 D, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, according to DE 19628705) was dissolved under nitrogen in 32 mL of anhydrous dimethyl sulfoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D), add 6.03mL 1,4-diaminobutane. After stirring at 40 for 17 hours, the reaction mixture was added to 150 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C, washed with 40 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol, and collected by centrifugation. The crude product was dissolved in 80 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of isolated product was 52%. the

Example 7.1 (d) Synthesis of Aldehyde HES10/0.7

150 mg of 4-formylbenzoic acid and 230 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 10 mL of N,N-dimethylformamide (peptide synthesis grade, Biosolve, Valkenswaard, NL), and 204 μL of N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added. After 30 min incubation at 21 °C, 1 g Amino HES10/0.7 (synthesized as described in 1.3) was added. After shaking at 22°C for 19 hours, the reaction mixture was added to 84 mL of ice-cold 2-propanol. The precipitated product was collected by centrifugation at 4°C, redissolved in 50 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of the isolated product was 83%. the

Example 7.1 (e) Synthesis of Amino HES30/0.4

Take 5 g of oxo-HES30/0.4 (MW=30000D, DS=0.4, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, using the molar ratio of the components according to DE 19628705A1) at 80°C under vacuum overnight, then dissolve in To 28 mL of anhydrous dimethylsulfoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added 1.67 mL of 1,4-diaminobutane. After stirring at 40°C for 17 hours, the reaction mixture was added to 175 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C. The crude product was dissolved in 40 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, PerbioSciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of isolated product was not determined. the

Synthesis of embodiment 7.1 (f) aldehyde group HES30/0.4

130 mg of 4-formylbenzoic acid and 153 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 36 mL of N,N-dimethylformamide (peptide synthesis Grade, Biosolve, Valkenswaard, NL), 110 μL of N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added. After incubation at 21°C for 30 min, 2.61 g Amino HES30/0.4 (synthesized as described in 1.5) was added. After shaking at 22°C for 22.5 hours, the reaction mixture was added to 160 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C and washed with an ice-cold 1:1 (v/v) mixture of acetone and ethanol. After centrifugation, the pellet was dissolved in 30 mL of water, dialyzed against water for 1 day (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of the isolated product was 81%. the

Embodiment 7.1 (g) synthesis of amino HES30/0.7

Take 5 g of oxo-HES30/0.7 (MW = 30000D, DS = 0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, using the composition molar ratio according to DE 19628705A1) at 80 ° C under vacuum overnight, then dissolved in To 28 mL of anhydrous dimethylsulfoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added 1.67 mL of 1,4-diaminobutane. After stirring at 40°C for 17 hours, the reaction mixture was added to 175 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C. The crude product was dissolved in 40 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, PerbioSciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of isolated product was not determined. the

Embodiment 7.1 (h) Synthesis of aldehyde group HES30/0.7

Take 122 mg 4-formylbenzoic acid and 144 mg 1-hydroxy-H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) dissolved in 34 mL N,N-dimethylformamide (peptide synthesis Grade, Biosolve, Valkenswaard, NL), 103 μL of N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added. After incubation for 30 min at 21°C, 2.46 g Amino HES30/0.7 (synthesized as described in 1.7) was added. After shaking at 22°C for 22.5 hours, the reaction mixture was added to 160 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C and washed with an ice-cold 1:1 (v/v) mixture of acetone and ethanol. After centrifugation, the pellet was dissolved in 30 mL of water, dialyzed against water for 1 day (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of the isolated product was 87%. the

Example 7.1 (i) Synthesis of Amino HES50/0.7

Take 6.09 g of oxo-HES50/0.7 (MW=50000D, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D, adopting molar ratios of components according to DE 19628705A1) heated at 80° C. under vacuum overnight, then dissolved under nitrogen. To 32 mL of anhydrous dimethylsulfoxide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added 1.22 mL of 1,4-diaminobutane. After stirring at 40° C. for 17 hours, the reaction mixture was added to 150 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4°C. Wash with an ice-cold 1:1 (v/v) mixture of acetone and ethanol and collect by centrifugation. The crude product was dissolved in 80 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of the isolated product was 82%. the

Example 7.1 (i) Synthesis of Aldehyde HES50/0.7

125 mg of 4-formylbenzoic acid and 174 mg of 1-hydroxy-1H-benzotriazole (both from Aldrich, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 38 mL of N,N-dimethylformamide (peptide synthesis Grade, Biosolve, Valkenswaard, NL), 155 μL of N,N'-diisopropylcarbodiimide (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) was added. After incubation for 30 min at 21°C, 3.8 g Amino HES50/0.7 (synthesized as described in 1.9) was added. After shaking at 22°C for 19 hours, the reaction mixture was added to 160 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation at 4 °C, redissolved in 20 mL of N,N-dimethylformamide, and precipitated with 80 mL of an ice-cold 1:1 (v/v) mixture of acetone and ethanol as described above. After centrifugation, the pellet was dissolved in 50 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tubing, cut-off value 3.5 kD, Perbio Sciences Deutschland GmbH, Bonn, D), and then lyophilized. The yield of isolated product was 77%. the

Example 7.2 Synthesis of HES-G-CSF conjugates by reductive amination

Example 7.2(a) Buffer Exchange A:

Take 33 mL of 0.454 mg/mL hG-CSF (XM02, BioGeneriX AG, Mannheim, D) in 10 mM sodium acetate solution, 50 mg/mL sorbitol and 0.004% Tween 80, pH 4.0, at 0 °C, using a Vivaspin 15R concentrator ( VS15RH11, 5KD MWCO, Vivascience AG, Hannover, D) was concentrated to 4 mL by diafiltration and diluted to 15 mL with 0.1 M sodium acetate buffer, pH 5.0. This diafiltration was repeated 2 times. The final concentration in this final diafiltration step was 3 mg/mL. the

Example 7.2(b) Reaction of hG-CSF with Aldehyde HES Derivatives of Examples 7.1(b), 7.1(d) and 7.1(j)

After buffer exchange into 0.1M sodium acetate buffer, pH 5.0 (as described in 7.2(a) above), to 1.67 mL of hG-C SF solution was added 1.67 mL of HES-derivative and 1.67 mL of 60 mM cyanoborohydro Sodium in the same buffer, this solution was incubated at 4°C for 15.5 hours. All solutions were cooled to 0°C before mixing. the

The following final HES concentrations were used:

39.4 mg/mL HES derivative prepared according to Examples 7.1(b) and 7.1(d). the

197 mg/mL of the HES derivative prepared according to Example 7.1(j). the

197mg/mL HES50/0.7 (MW=50000D, DS=0.7, Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D) was used as a reaction control. the

The reaction mixture was analyzed by gel electrophoresis (see Figure 17). the

Example 7.2(c) Buffer Exchange B:

Take 20 mL of 0.454 mg/mL hG-CSF (XM02, BioGeneriX AG, Mannheim, D) in 10 mM sodium acetate solution, 50 mg/mL sorbitol and 0.004% Tween 80, pH 4.0, at 15 °C, using a Vivaspin 15R concentrator ( VS15RH11, 5KD MWCO, Vivascience AG, Hannover, D) was concentrated to 4 mL by diafiltration and diluted to 15 mL with 0.1 M sodium acetate buffer, pH 5.0. This diafiltration was repeated 2 times. The final concentration in this final diafiltration step was 1.5 mg/mL. the

Example 7.2(b) Reaction of hG-CSF with the Aldehyde HES Derivatives of Examples 7.1(f) and 7.1(h)

After buffer exchange into 0.1M sodium acetate buffer, pH 5.0 (as described in 7.2(c) above), to 3.3 mL hG-CSF solution was added 3.3 mL 789 mg HES-derivative and 3.3 mL 60 mM cyanoborohydro Sodium in the same buffer, this solution was incubated at 4°C for 30 hours. All solutions were cooled to 0°C before mixing. the

A sample was taken after 17 hours for use as a reaction control. The reaction mixture was analyzed by gel electrophoresis (see Figure 18). the

Example 7.3 Synthesis of HES-GCFS conjugates by N,N'-succinimidyl carbonate coupling

Example 7.3(a) Synthesis of G-CSF conjugates through the reaction of hydroxyethyl starch with reactive ester groups and G-CSF

400 mg of oxo-HES 10/0.7 (prepared by Supramol Parenteral Colloids GmbH, Rosbach-Rodheim, D; degree of oxidation of oxo-HES 95% according to DE 19628705A1) was dissolved in 1 ml of anhydrous DMF. To this solution was added 21 mg of N,N'-disuccinimidyl carbonate, and the mixture was stirred at room temperature for 2 hours. The resulting solution had a reactive HES concentration of 40% by weight. the

The G-CSF solution (Strathmann BiotecAG, Hamburg, D) with a concentration of about 0.5 mg G-CSF/ml was concentrated to a concentration of 10 mg/ml by ultracentrifugation with a cut-off value of 100 kD in a cooling centrifuge. the

To 0.5 ml of this concentrated G-CSF solution was added 180 μl of sodium bicarbonate solution. Then the reactive HES solution was added dropwise to the protein solution in 3 portions (100 μl each time) until the reaction was completed after about 30 minutes. The total molar ratio of reactive HES:G-CSF is therefore about 50:1. The pH of the mixture was then adjusted to 4.0 using 0.1N HCl. the

HPGPC analysis (High Performance Gel Permeation Chromatography) gave yields in excess of 95%. Unreacted G-CSF was not detected. The results are shown in Figure 19. the

Example 7.4 In vitro assay

Mitogenicity of G-CSF variants on mouse NFS-60 cells

G-CSF is known to have specific effects on the proliferation, differentiation and activation of hematopoietic cells of the neutrophil lineage. The mitogenic ability of G-CSF variants was tested using mouse NFS-60 cells (N. Shirafuji et al., Exp. Hematol. 1989, 17, 116-119). Cells were cultured in RPMI medium containing 10% fetal bovine serum (Gibco INVITROGEN GmbH, Gibco INVITROGEN GmbH, Karlsruhe, D) were grown, harvested by centrifugation, washed, and split in 24-well plates at 100,000 cells per well. Cells were adapted for 1 hour at 37°C in RPMI medium without WEHI-3B conditioned medium, and then G-CSF growth factor samples diluted in the same medium were added. NFS-60 cells were exposed to purified G-CSF variants for 3 days at 37°C (purification according to Example 5.3, 5.4, protein quantification according to Example 5.5(a)):

均来自Amgen， 

Both from Amgen,

"HES-GCFS10/0.4 conjugate" was prepared according to Example 7.2(b),

"HES-GCFS10/0.7 conjugate" was prepared according to Example 7.2(b),

"HES-GCFS30/0.4 conjugate" was prepared according to Example 7.2(d),

"HES-GCFS30/0.7 conjugate" was prepared according to Example 7.2(d),

"HES-GCFS50/0.7 conjugate" was prepared according to Example 7.2(b),

"HES-GCFS10/0.7 conjugate (Supramol)" was prepared according to Example 7.3 (a),

"Pseudo-incubation" (=reaction control, 197mg/ml HES50/0.7, MW 50000D, DS7, Supramol Parenteral Colloids GmbH, Rosbach Rodheim, Germany),

Cells were then counted electronically (Casy TT cell counter, Scharfe System, Reutlingen, D). The results are shown in Table 2 and Fig. 20 . In all cases, the protein content shown in Table 2 and Figure 20 represents the G-CSF content of the conjugate only and is based on the concentration determined by GlycoThera. As can be seen from Figure 20, different G-CSF variants (2.5-250 pg/ml) could stimulate an increase in cell number after 3 days compared to the medium without growth factor addition. All variants reached the same maximal stimulation level at a concentration of 250 pg/ml. the

Table 2: Proliferation of mouse NFS-60 cells induced by G-CSF variants

Concentration [pg/ml] 0 2.5 2.8 5 5.7 10 11.3 25 28.4 50 56.7 250 283.5 Neupogen 0.44 0.86 the 1.20 the 1.69 the 2.33 the 2.49 the 2.41 the HES-GCSF 10/0.7 0.44 0.72 the 0.93 the 1.44 the 2.14 the 2.41 the 2.41 the HES-GCSF 10/0.4 0.44 0.72 the 0.97 the 1.40 the 2.17 the 2.67 the 2.75 the HES-GCSF 50/0.7 0.44 0.62 the 0.70 the 0.97 the 1.68 the 2.15 the 2.32 the Pseudo-incubation 0.44 0.85 the 1.31 the 1.91 the 2.38 the 2.47 the 2.41 the HES-GCSF 30/0.4 0.44 0.82 the 1.21 the 1.62 the 2.28 the 2.50 the 2.60 the HES-GCSF 30/0.7 0.44 0.80 the 1.09 the 1.66 the 2.20 the 2.35 the 2.44 the Neulasta 0.44 0.63 the 0.80 the 1.12 the 1.83 the 2.25 the 2.33 the HES-GCSF 10/0.7 (Supramol) 0.44 the 0.73 the 1.13 the 1.58 the 2.24 the 2.48 the 2.46

Example 7.5 In vivo biological effects of hG-CSF conjugates in rats

大鼠(7周大)，Charles RiverDeutschland GmbH，Sanghofer Weg 7，D-97633Sulzfeld]随机分成每组5只。适应7天后，淘汰状态较差的大鼠，换成备用动物。大鼠抵达时的体重为181-203g。  Upon arrival of rats [male

Rats (7 weeks old), Charles RiverDeutschland GmbH, Sanghofer Weg 7, D-97633 Sulzfeld] were randomly divided into groups of 5. After 7 days of adaptation, rats in poor condition were eliminated and replaced with spare animals. Rats weighed 181-203 g on arrival.

Five rats randomly selected in each group were intravenously administered the following unconjugated or conjugated G-CSF samples (purified according to Example 5.3, 5.4, and protein quantified according to Example 5.5(a)), per kg body weight Administration of 100 μg protein (injection speed 15 sec/dose, vehicle: 5 ml PBS/kg body weight):

均来自Amgen， 

Both from Amgen,

"HES-GCFS10/0.4 conjugate" (10/0.4) was prepared according to Example 7.2(b),

"HES-GCFS10/0.7 conjugate" (10/0.7) was prepared according to Example 7.2 (b),

"HES-GCFS30/0.4 conjugate" (30/0.4) was prepared according to Example 7.2(d),

"HES-GCFS30/0.7 conjugate" (30/0.7) was prepared according to Example 7.2(d),

"HES-GCFS50/0.7 conjugate" (50/0.7) was prepared according to Example 7.2 (b),

"HES-GCFS10/0.7 conjugate (Supramol)" (S10-0.7) was prepared according to Example 7.3(a),

"sham incubation" (=reaction control, 197mg/ml HES50/0.7, MW 50000D, DS7, Supramol Parenteral Colloids GmbH, Rosbach Rodheim, Germany), and

Vector control

Under light ether anesthesia, blood samples of approximately 200 μL of LEDTA whole blood were drawn from the retroocular venous plexus of all animals. All animals fasted overnight were bled once in the morning 5 days before the test. On days 1 to 8 of the test, blood was collected twice a day with an interval of 12 hours. The first blood sample on the first day was taken before administration of the G-CSF/GCSF-conjugate. the

White blood cell (WBC) counts were performed with a Bayer ADVIA ^™ 120 (Fernwald, Germany). The results are shown in Fig. 21 .

Claims (39)

1. method for preparing the conjugate that comprises protein and polymer derivant; Wherein said polymer derivant obtains through in polymer, introducing at least one functional group A, and this polymer is that hydroxyalkyl starch and protein are granulocyte colony-stimulating factor, and this method comprises at least one functional group A and proteinic at least one Z of the functional group reaction that makes said polymer derivant; Form covalently bound thus; Wherein Z is amino, and A is selected from aldehyde radical, ketone group or hemiacetal group

This method also comprises by polymer and the reaction of difunctional compound at least introduces A in polymer; To produce said polymer derivant; This is one of them functional group and the polymer reaction of difunctional compound at least; And another functional group is aldehyde radical, ketone group or hemiacetal group at least, or further chemical modification can produce the functional group of aldehyde radical, ketone group or hemiacetal group

Wherein said polymer derivant and proteinic reaction are reductive amination.

2. according to the process of claim 1 wherein that said hydroxyalkyl starch is a hetastarch.

3. according to the method for claim 2, the molecular weight of wherein said hetastarch is 2 to 200kD.

4. according to each method in the claim 1 to 3; This method also comprises by polymer and the M of the functional group reaction of difunctional compound at least; Produce polymer derivant, this at least difunctional compound comprise also at least that another is aldehyde radical, the Q of functional group of ketone group or hemiacetal group A.

5. according to the method for claim 4, wherein M comprises amino.

6. according to each method in the claim 1 to 3; This method also comprises by polymer and the M of the functional group reaction of difunctional compound at least; Produce polymer derivant; This at least difunctional compound also comprise the Q of other functional group that at least one is not aldehyde radical, ketone group or hemiacetal group, this method also comprises by the reaction of the Q of functional group and at least a suitable compound, produces the polymer derivant that comprises aldehyde radical, ketone group or hemiacetal group A.

7. according to the method for claim 5, wherein M and Q comprise amino.

8. according to the method for claim 6, wherein said at least a suitable compound comprises carboxyl and aldehyde radical, ketone group or hemiacetal group.

9. according to Claim 8 method, wherein said at least a suitable compound is formoxyl benzoic acid or 4-(4-formoxyl-3,5-dimethoxy phenoxy group) butanoic acid.

10. according to the method for claim 6, wherein M comprises amino, and Q comprises beta-hydroxy amino.

11. according to the method for claim 10, wherein said polymer is in the reducing end of its oxidation and the M of the functional group reaction of difunctional compound at least.

12. according to the method for claim 10, it also comprises oxidation of beta-hydroxyl amino, produces aldehyde radical.

13. according to the method for claim 12, wherein said oxidation reaction uses periodate to carry out.

14. according to the process of claim 1 wherein that reductive amination is at NaCNBH ₃Existence under carry out.

15. according to the process of claim 1 wherein that reductive amination is pH7 or following carrying out.

16. according to the method for claim 15, wherein this pH be 6 or below.

17. according to the process of claim 1 wherein that reductive amination carries out under 0 to 25 ℃ temperature.

18. according to the process of claim 1 wherein that reductive amination carries out in aqueous medium.

19. conjugate that can make according to each method in the claim 1 to 18.

20. a conjugate that comprises protein and polymer or derivatives thereof, wherein this polymer is a hydroxyalkyl starch, and protein is granulocyte colony-stimulating factor, its have as shown in the formula structure:

And/or

R wherein ₁, R ₂And R ₃Independent respectively is hydrogen or hydroxy alkyl, hydroxyaryl, hydroxyl aralkyl or the hydroxyl alkaryl with 2 to 10 carbon atoms, and

Wherein L is optional substituted straight chain, branch and/or cyclic hydrocarbon residue, chooses wantonly to comprise at least one hetero atom, has 1 to 60 carbon atom.

21. according to the conjugate of claim 20, wherein L is CH ₂

22. a conjugate that comprises protein and polymer or derivatives thereof, wherein this polymer is a hydroxyalkyl starch, and protein is granulocyte colony-stimulating factor, its have as shown in the formula structure:

R wherein ₁, R ₂And R ₃Independent respectively is hydrogen or hydroxy alkyl, hydroxyaryl, hydroxyl aralkyl or the hydroxyl alkaryl with 2 to 10 carbon atoms, and

L wherein ₁And L ₂Independent respectively is optional substituted straight chain, branch and/or cyclic hydrocarbon residue, chooses wantonly to comprise at least one hetero atom, comprises alkyl, aryl, the assorted alkyl of aralkyl and/or heteroarylalkyl part, and said residue has 1 to 60 carbon atom, and

Wherein D serves as reasons and connects L ₁Appropriate functional group F ₂Be connected L ₂Appropriate functional group F ₃Formed covalently bound, F wherein ₂Be selected from:

The two keys of-C-C-or C-C-triple bond or aromatic series C-C-key;

-mercapto or hydroxyl;

-alkyl sulfonic acid hydrazides, aryl sulfonic acid hydrazides;

-1, the 2-dihydroxylic alcohols;

-1,2-amino-thio-alcohol;

-azide;

-1, the 2-alkamine;

-amino-NH ₂, or aminoalkyl, aminoaryl, amino aralkyl or alkaryl are amino;

-hydroxyl amino-O-NH ₂, or hydroxy alkyl is amino, hydroxyaryl is amino, hydroxyl aralkyl is amino or the hydroxyl alkaryl is amino;

-alkoxy amino, aryloxy group are amino, aralkoxy is amino or aryloxy alkyl is amino, and it respectively comprises structural units-NH-O-;

-have carbonyl residue-Q-C (=G)-M, wherein G is O or S, M is:

---OH or-SH;

--alkyl oxy, aryloxy, aralkyl oxy or alkaryl oxygen base;

--alkyl sulfenyl, artyl sulfo, aromatic alkyl sulfurio or alkaryl sulfenyl;

--alkyl-carbonyl oxygen base, aryl carbonyl oxygen base, aromatic alkyl carbonyl oxygen base, alkaryl ketonic oxygen base;

--activatory ester;

Wherein Q does not exist or is NH or hetero atom;

--NH-NH ₂Or-NH-NH-;

--NO ₂；

-itrile group;

-carbonyl;

-carboxyl;

--the N=C=O group or-the N=C=S group;

-vinyl halide group or TFMS root;

--C≡C-H；

--(C=NH ₂C1)-the O alkyl

--(C=O)-CH ₂-Hal group, wherein Hal is Cl, Br or I;

--CH＝CH-SO ₂-；

-comprise the disulphide group of structure-S-S-;

-group

-group

, F wherein ₃Be can with F ₂Form the functional group of chemical bond.

23. according to the conjugate of claim 22, wherein L ₁For-(CH ₂) n-, wherein n=2,3,4,5,6,7,8,9,10.

24. according to the conjugate of claim 22 or 23, wherein L ₂Comprise optional suitable substituted aryl moiety.

25. according to the conjugate of claim 24, wherein L ₂Be C ₆H ₄

26. according to the conjugate of claim 22, wherein F ₂Comprise-the NH-part.

27. according to the conjugate of claim 22, wherein F ₃Comprise-(C=G)-part.

28. according to the conjugate of claim 22, wherein F ₂Comprise amino, and F wherein ₃Comprise-(C=G)-the G-part.

29. according to the conjugate of claim 22, wherein F ₂Comprise-the NH-part, and F wherein ₃Comprise-(C=O)-and the O part, D is an amido link.

30. according to each conjugate in the claim 20 to 23, wherein said hydroxyalkyl starch is a hetastarch.

31. according to the conjugate of claim 30, the molecular weight of wherein said hetastarch is 2 to 200kD.

32. a pharmaceutical composition, its comprise the treatment effective dose according to each conjugate in the claim 20 to 31.

33. according to the pharmaceutical composition of claim 32, it also comprises at least a pharmaceutically acceptable diluent, adjuvant or carrier.

34. a pharmaceutical composition, it comprises the conjugate according to claim 19 of treating effective dose.

35. according to the pharmaceutical composition of claim 34, it also comprises at least a pharmaceutically acceptable diluent, adjuvant or carrier.

36. be used for treating the purposes of the medicine that hemopoietic or immunologic function reduce in preparation according to each conjugate in the claim 20 to 31.

37. according to the purposes of claim 36, wherein said hemopoietic or immunologic function reduce and are caused by chemotherapy, X-ray therapy, infectious disease, severe chronic neutrophilic granulocytopenia or leukemia.

38. be used for treating the purposes of the medicine that hemopoietic or immunologic function reduce in preparation according to the conjugate of claim 19.

39. according to the purposes of claim 38, wherein said hemopoietic or immunologic function reduce and are caused by chemotherapy, X-ray therapy, infectious disease, severe chronic neutrophilic granulocytopenia or leukemia.

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